nitrophenols and 4-nitrophenyl-acetate

Topics: Amino Acid Sequence; Binding Sites; Esterases; Humans; Nitrophenols; Pharmaceutical Preparations; Protein Binding; Serum Albumin

To characterize the relationships between human plasma irinotecan carboxylesterase-converting enzyme activity, caboxylesterase-mediated hydrolysis of p-nitrophenyl acetate (pNPA), and the butyrylcholinesterase-mediated hydrolysis of butyrylthiocholine in human plasma and to test the ability of these in vitro tests to predict the variability in SN-38 pharmacokinetics in adult patients during a prolonged infusion of irinotecan.. Individual plasma-converting enzyme activity was measured in 20 adult cancer patients participating in a pharmacokinetic and phase I clinical trial of a prolonged 96-h intravenous infusion of irinotecan. The pNPA and butyrylthiocholine hydrolysis in patient plasma was also assayed.. The irinotecan carboxylesterase-converting enzyme in human plasma had a Vmax of 89.9 +/- 22.7 pmol/h per ml plasma and a Km of 207 +/- 56 microM (mean +/- SD, n = 3). The mean value of the specific activity of this enzyme in 20 adult cancer patients was 10.08 +/- 2.96 pmol/h per ml plasma ranging from 5.43 to 15.39 pmol/h per ml. The area-under-the-concentration-versus time curve (AUC) ratio of SN-38 to irinotecan (AUCSN-38/AUCCPT-11) was used to assess the relative SN-38 exposure to the active metabolite in individual patients. Pharmacokinetic variations in the relative exposure to SN-38 did not correlate with the measured carboxylesterase-converting enzyme activity nor with plasma butyrylcholinesterase activity in our patient population. However, it did correlate with the measured pNPA hydrolysis activity in patient plasma (r2 = 0.350, P = 0.0124, n = 18).. Determination of patient plasma pNPA hydrolysis activity may have utility in predicting SN-38 pharmacokinetics during prolonged infusions of irinotecan.

Topics: Antineoplastic Agents, Phytogenic; Area Under Curve; Butyrylcholinesterase; Butyrylthiocholine; Camptothecin; Carboxylic Ester Hydrolases; Humans; Hydrolysis; Irinotecan; Nitrophenols; Predictive Value of Tests

A total of 164 blood samples from 16 clinically healthy bottlenose dolphins (Tursiops truncatus), were obtained from an aquarium in Spain between 2019 and 2020, as part of their preventive medicine protocol. In addition to conventional haematological and biochemical analyses, plasmatic B-esterase activities were characterised to determine the potential application of such analyses in wild counterparts. The hydrolysis rates for the substrates of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and carboxylesterase (CE) activity in plasma were measured, the last using two commercial substrates, p-nitrophenyl acetate (pNPA) and p-nitrophenyl butyrate (pNPB). Activity rates (mean ± SEM in nmol/min/mL plasma) were (in descending order): AChE (125.6 ± 3.8), pNPB-CE (65.0 ± 2.2), pNPA-CE (49.7 ± 1.1) and BuChE (12.8 ± 1.3). These values for dolphins are reported in here for the first time in this species. Additionally, the in vitro sensitivity of two B-esterases (AChE and pNPB-CE) to chemicals of environmental concern was determined, and the protective role of plasmatic albumin assessed. Out of the B-esterases measured in plasma of dolphin, AChE activity was more responsive in vitro to pesticides, while CEs had a low response to plastic additives, likely due to the protective presence of albumin. However, the clear in vitro interaction of these environmental chemicals with purified AChE from electric eels and recombinant human hCEs (hCE1 and hCE2) and albumin, predicts their impact in other tissues that require in vivo validation. A relationship between esterase-like activities and health parameters in terrestrial mammals has already been established. Thus, B-esterase measures could be easily included in marine mammal health assessment protocols for dolphins as well, once the relationship between these measures and the animal's fitness has been established.

Topics: Acetylcholinesterase; Albumins; Animals; Biomarkers; Bottle-Nosed Dolphin; Butyrylcholinesterase; Carboxylesterase; Esterases; Humans; Nitrophenols; Pesticides; Plastics; Xenobiotics

Serum albumin possesses esterase and pseudo-esterase activities towards a number of endogenous and exogenous substrates, but the mechanism of interaction of various esters and other compounds with albumin is still unclear. In the present study, proton nuclear magnetic resonance (

Topics: Binding Sites; Biocatalysis; Crystallization; Esterases; Hydrolysis; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Nitrophenols; Phenylpropionates; Protein Binding; Proton Magnetic Resonance Spectroscopy; Serum Albumin, Bovine; Serum Albumin, Human

Hydrolytic reactions constitute an important pathway of drug metabolism and a significant route of prodrug activation. Many ophthalmic drugs and prodrugs contain ester groups that greatly enhance their permeation across several hydrophobic barriers in the eye before the drugs are either metabolized or released, respectively,

Topics: Animals; Carboxylesterase; Eye; Female; Humans; Hydrolysis; Male; Nitrophenols; Prodrugs; Proteomics; Rabbits; Substrate Specificity; Swine

To increase milk production, antibiotics are administered to animals to provide weight gain and to prevent or treat diseases. The inappropriate use of these substances can lead to antibiotic resistance and allergic reactions and toxic effects to milk consumers. We describe the development of a simple, fast, portable, and low-cost microfluidic paper-based analytical device (μPAD) to quantify sulfonamides in milk using the inhibition of the colorimetric reaction between carbonic anhydrase (CA) and 4-nitrophenyl acetate. The main advantages presented by the μPAD include reproducible batch production, simple application, and precise analysis without previous treatment. The µPAD displayed good linearity (R

Topics: Animals; Carbonic Anhydrases; Cattle; Colorimetry; Hydrogen-Ion Concentration; Microfluidic Analytical Techniques; Milk; Nitrophenols; Paper; Sulfonamides

Human carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) are serine-esterase enzymes catalyzing the hydrolysis of many compounds containing esters, amides, thioesters, or acetyl groups. This study aimed to investigate the presence, kinetic parameters, and inhibition of CES1, CES2, and AADAC in A549, H460, and H727 pulmonary cells in both living cells and S9 fractions.. The p-nitrophenyl acetate (pNPA) and 4-methylumbelliferyl acetate (4-MUA) were used as non-selective esterase substrates, whereas phenacetin as selective AADAC substrate. CESs activities were also investigated in living cells by cellular bioimaging using selective fluorescent probes.. AADAC gene was detected in A549 and H460 cells; nevertheless, arylesterase activity was not found in relative S9 fractions. Besides, CES1 and CES2 were expressed to a different extent by all lung cells, and enzymatic activities were quite overlapping each other. All enzymes exhibited a typical Michaelis-Menten saturation curve and, regarding 4-MUA, similar K. These findings add information to esterase knowledge in pulmonary cells that could be used as in vitro models for toxicological and pharmacological studies.

Topics: A549 Cells; Carboxylesterase; Carboxylic Ester Hydrolases; Cell Line; Esterases; Humans; Hydrolysis; Lung; Microsomes, Liver; Nitrophenols; Phenacetin; Substrate Specificity; Umbelliferones

Mammalian paraoxonase-1 hydrolyses a very broad spectrum of esters such as certain drugs and xenobiotics. The aim of this study was to determine whether carbamates influence the activity of recombinant PON1 (rePON1). Carbamates were selected having a variety of applications: bambuterol and physostigmine are drugs, carbofuran is used as a pesticide, while Ro 02-0683 is diagnostic reagent. All the selected carbamates reduced the arylesterase activity of rePON1 towards the substrate

Topics: Acetates; Aryldialkylphosphatase; Carbamates; Carbofuran; Carboxylic Ester Hydrolases; Humans; Models, Molecular; Nitrophenols; Phenols; Terbutaline

Esterases catalyze the hydrolysis of ester bonds in fatty acid esters with short-chain acyl groups. In the present study, thirty-seven bacterial isolates were isolated from soil contaminated with waste cooking oil, dairy waste etc. from Shimla and Solan district of H.P. Out of 37 isolates, the isolate RL-1, which gave maximum activity, was identified as Bacillus licheniformis MH061919. The optimization of various production parameters resulted in maximum activity at inoculum age of 24 h and inoculum size of 1.5% (v/v). Esterase gave considerable activity in production medium containing sodium chloride (0.5 % w/v), galactose (1%, w/v), coconut oil (2.0%, v/v) and beef extract (0.3%, w/v) at a temperature of 45℃ and pH 8.5.The enzyme production was enhanced by 3-fold after optimization of production parameters. Further, on optimizing reaction conditions, enzyme gave maximum activity at a temperature of 45℃ and pH 8.5. The para-nitrophenyl acetate (p-NPA) was found to be optimum substrate and metal ions and detergents have inhibitory effect on esterase activity.

Topics: Bacillus; Coconut Oil; Culture Media; Culture Techniques; Esterases; Galactose; Hydrogen-Ion Concentration; Nitrophenols; Red Meat; Sodium Chloride; Temperature; Tissue Extracts

Self-assembling peptides are attracting wide interest as biodegradable building blocks to achieve functional nanomaterials that do not persist in the environment. Amongst the many applications, biocatalysis is gaining momentum, although a clear structure-to-activity relationship is still lacking. This work applied emerging design rules to the heterochiral octapeptide sequence His-Leu-

Topics: Biocatalysis; Esterases; Hydrogels; Nanofibers; Nitrophenols; Peptide Fragments

Three novel binuclear copper(ii) complexes with reduced l-serine Schiff bases were synthesized and their structures were analyzed with single-crystal X-ray diffraction and DFT calculations. The crystal data revealed that all of these binuclear complexes are chiral. Both 5-halogenated (bromo- and chloro-) binuclear complexes exhibit right-handed helix structural character. Interestingly, the 5-methyl-containing analogue has a two-dimensional pore structure. In this paper, the esterolysis reactivity of the as-prepared complexes shows that in the hydrolysis of p-nitrophenyl acetate (PNPA) these three complexes provide 26, 18, 40-fold rate acceleration as compared to the spontaneous hydrolysis of PNPA at pH 7.0, respectively. Under selected conditions, in excess buffered aqueous solution a rate enhancement by three orders of magnitude was observed for the catalytic hydrolysis of another carboxylic ester, p-nitrophenyl picolinate (PNPP). These complexes efficiently promoted PNPP hydrolysis in a micellar solution of cetyltrimethylammonium bromide (CTAB), giving rise to a rate enhancement in excess of four orders of magnitude, which is approximately 2.0-3.2 times higher than that in the buffer.

Topics: Biomimetic Materials; Carboxylic Ester Hydrolases; Coordination Complexes; Copper; Density Functional Theory; Esters; Hydrolysis; Models, Molecular; Molecular Structure; Nitrophenols; Oxidation-Reduction; Schiff Bases; Serine

Studies with acetylcholinesterase (AChE) inhibited by organophosphorus (OP) compounds with two chiral centers can serve as models or surrogates for understanding the rate, orientation, and postinhibitory mechanisms by the nerve agent soman that possesses dual phosphorus and carbon chiral centers. In the current approach, stereoisomers of

Topics: Acetylcholinesterase; Animals; Cholinesterase Inhibitors; Esters; Mice; Molecular Structure; Nitrophenols; Organophosphorus Compounds; Sulfhydryl Compounds

The metA (Rv3341) gene from Mycobacterium tuberculosis H37Rv strain encodes a homoserine-acetyltransferase (HAT) enzyme, also called MetA. This enzyme plays a key role in the biosynthetic pathway of methionine and is a potential target for the development of antimicrobial drugs. Purified MetA showed 40 kDa molecular mass on SDS-PAGE. Manual docking was performed with substrates acetyl-CoA, l-homoserine, and p-nitrophenylacetate using crystal structure coordinates of MetA (PDB ID 6PUX) from M. tuberculosis. Multiple sequence alignment indicated that catalytic triad residues Ser157, Asp320, His350 were conserved across species in acetyltransferases, esterases, and hydrolases. As a conserved pentapeptide, GXSMG belongs to α/β hydrolase superfamily and it shares similarity with esterases and hydrolases from different sources. Hydrolase activity of MetA was tested using (PNPA), N-acetylglycine, N-acetylmethionine and Phe-Gly as substrate. LC-MS confirmed that MetA possessed HAT activity, but no homoserine-succinyltransferase (HST) and serine-acetyltransferase (SAT) activities. Replacing acetyl-CoA with PNPA as acetyl group donor showed a drastic reduction in transferase activity, arising due to the interaction of R227 of the enzyme with PNPA. This could prevent the binding of the second substrate in the right orientation and results in the preferential transfer of the acetyl group to water, thus exhibiting hydrolase rather than transferase activity. In this paper, we report that MetA has both transferase and hydrolase activity depending on the correct orientation of the second substrate and the availability of the amino acids involved in enzyme-substrate interaction.

Topics: Acetyl Coenzyme A; Acetyltransferases; Binding Sites; Catalytic Domain; Crystallography, X-Ray; Hydrolases; Kinetics; Molecular Docking Simulation; Mycobacterium tuberculosis; Nitrophenols; Phylogeny; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity

Esterases are a large family of enzymes with wide applications in the industry. However, all esterases originated from natural sources, limiting their use in harsh environments or newly- emerged reactions. In this study, we designed a new esterase to develop a new protocol to satisfy the needs for better biocatalysts. The ideal spatial conformation of the serine catalytic triad and the oxygen anion hole at the substrate-binding site was constructed by quantum mechanical calculation. The catalytic triad and oxygen anion holes were then embedded in the protein scaffold using the new enzyme protocol in Rosetta 3. The design results were subsequently evaluated, and optimized designs were used for expression and purification. The designed esterase had significant lytic activities towards p-nitrophenyl acetate, which was confirmed by point mutations. Thus, this study developed a new protocol to obtain novel enzymes that may be useful in unforgiving environments or novel reactions.

Topics: Catalytic Domain; Databases, Protein; Esterases; Hydrolysis; Mutation; Nitrophenols; Oxygen; Protein Engineering; Quantum Theory; Recombinant Proteins

An enzymatic hydrolysis of p-nitrophenyl acetate with carboxylesterase was analyzed by capillary electrophoresis/dynamic frontal analysis (CE/DFA). A plateau signal was expected with the anionic product of p-nitrophenol by the CE/DFA applying in-capillary reaction and the continuous CE resolution of the product from the substrate zone. However, the plateau height was not sufficient, and/or the plateau signal fluctuated and drifted. Therefore, a pressure assist was utilized in the CE/DFA to detect the product zone fast and to average the fluctuated plateau signal by mixing in a laminar flow. The plateau signal became relatively flat and its height was developed by the pressure-assisted capillary electrophoresis/dynamic frontal analysis (pCE/DFA). The plateau height was used for the Michaelis-Menten analysis, and a Michaelis-Menten constant was determined as KM = 0.83 mmol L-1. An enzyme inhibition was also examined with bis(p-nitrophenyl) phosphate by adding it in the separation buffer. The height of the plateau signal decreased by the inhibition, and a 50% inhibitory concentration was determined as IC50 = 0.79 μmol L-1. The values of KM and IC50 obtained in this study agreed well with the reported values. Since the proposed pCE/DFA includes electrophoretic migration of the substrate zone in a capillary, it is also noticed that the deactivation of the enzyme by ethanol on the preparation of the substrate solution can be avoided, as well as the exclusion of the inhibition by the product.

Topics: Carboxylesterase; Electrophoresis, Capillary; Hydrolysis; Kinetics; Nitrophenols

The design of interfaces that selectively react with molecules to transform them into compounds of industrial interest is an emerging area of research. An example of such reactions is the hydrolytic conversion of ester-based molecules to lipids and alcohols, which is of interest to the food, and pharmaceutical industries. In this study, a functional bio-interfaced layer was designed to hydrolyze 4-nitrophenyl acetate (pNPA) and Ricinus Communis (castor) oil rich in triglycerides using lipase b from Candida antarctica (CALB, EC 3.1.1.3). The attachment of CALB was performed via non-covalent immobilization over a polymer film of vertically aligned cylinders that resulted from the self-assembly of the di-block copolymer polystyrene-block-poly(4-vinyl pyridine) (PS-b-P4VP). This polymer-lipase model will serve as the groundwork for the design of further bioactive layers for separation applications requiring similar hydrolytic processes. Results from the fabricated functional bio-interfaced material include cylinders with featured pore size of 19 nm, d spacing of 34 nm, and ca. 40 nm of thickness. The polymer-enzyme layers were physically characterized using AFM, XPS, and FTIR. The immobilized enzyme was able to retain 91% of the initial enzymatic activity when using 4-nitrophenyl acetate (pNPA) and 78% when exposed to triglycerides from castor oil.

Topics: Candida; Castor Oil; Environmental Pollutants; Enzymes, Immobilized; Fungal Proteins; Humans; Hydrolysis; Lipase; Nitrophenols; Polystyrenes; Polyvinyls; Porosity; Ricinus; Triglycerides

Serum albumin, recognized mainly for its capacity to act as a carrier protein for many compounds, can also actively transform some organic molecules. As a starting point in this study, we consider esterase-like activity of bovine serum albumin (BSA) toward p-nitrophenyl acetate (p-NPA). Our results reveal that the reaction goes beyond ester hydrolysis step. In fact, the transformation product, p-nitrophenol (p-NP), becomes a substrate for further reaction with BSA in which its nitro group in subtracted and released in the form of HNO

Topics: Animals; Cattle; Electron Transport; Esterases; Histidine; Hydrolysis; Infrared Rays; Nitrophenols; Nitrous Acid; Serum Albumin, Bovine

Nanozymes provide comparative advantages over natural enzymes and conventional artificial enzymes for catalytic reactions. However, nanozymes are only suitable for limited types of reactions, whose catalytic principles are not yet fully revealed. Herein, a new nanozyme based on a bionic zeolitic imidazolate framework is proposed. Zeolitic imidazolate framework-8 (ZIF-8) possesses a similar geometric structure to that of the active center of human carbonic anhydrase II (hCAII) and exhibits catalytic performance analogous to that of the hCAII. The less imidazolate coordinated zinc cations on the external surface of ZIF-8 can act as Lewis acid sites, lowering the pKa of Zn-bound H2O molecules from 14 to 8.4, which facilitates the deprotonation of H2O molecules and generation of zinc-bound hydroxide nucleophiles. The esterase-like ZIF-8 nanozyme shows a similar affinity to p-nitrophenyl acetate compared with hCAII. The ZIF-8 nanozyme also promotes CO2 hydration and acetylthiocholine hydrolysis reaction, and a series of ZIFs are also found with intrinsic enzyme-like activities due to similar compositions and spatial structures. These results imply that the bionic nanoparticles can be developed to fabricate a new generation of nanozymes by mimicking the active sites of natural enzymes.

Topics: Acetylthiocholine; Biocompatible Materials; Carbon Dioxide; Carbonic Anhydrase II; Catalysis; Catalytic Domain; Humans; Hydrolysis; Imidazoles; Metal-Organic Frameworks; Nitrophenols; Zeolites

The main objectives of this study were to purify the glutathione S-transfereses (GSTs) and assess the effect of high doses of acrylamide (ACR) on male albino Wistar rat liver, kidney, testis and bran GST activities, and expression analysis of GST. ACR (50 mg/300 ml) was ingested for 40 days (20 doses) in drinking water on alternative days, on 40 day post ingestion the control and treated tissues were collected for GST purification by affinity column and biochemical characterization of GSTs by substrate specificities, and GST expression by immuno dot blots. In the analysis of the purified GSTs, we observed that liver GSTs were resolved in to three bands known as Yc, Yb and Ya; kidney GSTs were resolved in to two bands known as Yc and Ya; testis and brain GSTs were resolved as four bands known as Yc, Yb, Yβ and Yδ on 12.5% sodium dodecyl sulfate polyacrylamide gel (SDS PAGE). In the analysis of biochemical characterization, we observed a significant decrease (p < 0.05) in the specific activities of liver GST isoforms with the substrates 1-chloro 2,4-dinitrobenzene (CDNB), bromosulfophthalein (BSP), p-nitrophenyl acetate (pNPA), p-nitrobenzyl chloride (pNBC) and cumene hydroperoxide (CHP), but showed no activity with ethacrynic acid (ECA) and significant decrease (p < 0.05) in the specific activities of kidney GST isoforms with the substrates CDNB, pNPA, pNBC and CHP, but showed no activity with BSP and ECA, and a significant decrease (p < 0.05) in the specific activities of testis and brain GST isoforms with the substrates CDNB, BSP, pNPA, pNBC, ECA and CHP. In the analysis of immuno dot blots, we observed a decreased expression of liver, kidney, testis and brain GSTs. Through the affinity purification and biochemical characterization, we observed a tissue specific distribution of GSTs that is liver GSTs possess Yc, Yb and Ya sub units known as alpha (α) and mu (μ) class GSTs; kidney GSTs possess Yc and Ya sub units known as (α) alpha class GST; testis and brain GSTs possess Yc, Yb, Yβ and Yδ sub units known as alpha (α), mu (μ) and pi (π) class GSTs. Purification studies, biochemical characterization and immuno dot blot analysis were revealed the GSTs were sensitive to high doses of ACR and the high level exposure to ACR cause the damage of detoxification function of GST due to decreased expression and hence lead to cellular dysfunction of vital organs.

Topics: Acrylamide; Animals; Electrophoresis, Polyacrylamide Gel; Glutathione; Glutathione Transferase; Isoenzymes; Kidney; Liver; Male; Nitrobenzenes; Nitrophenols; Rats; Rats, Wistar; Substrate Specificity; Testis; Tissue Distribution

We developed an artificial hydrolase based on the symmetrical Pizza6 β-propeller protein for the metal-free hydrolysis of 4-nitrophenyl acetate and butyrate. Through site-specific mutagenesis and crystallisation studies, the catalytic mechanism was investigated and found to be dependent on a threonine-histidine dyad. The mutant with additional histidine residues generated the highest kcat values, forming a His-His-Thr triad and matched previously reported metalloenzymes. The highly symmetrical β-propeller artificial enzymes and their protein-metal complexes have potential to be utilised in bioinorganic and supramolecular chemistry, as well as being developed further into 2D/3D catalytic materials.

Topics: Amino Acid Sequence; Aspartic Acid; Butyrates; Catalysis; Copper; Histidine; Hydrolases; Hydrolysis; Kinetics; Mutagenesis, Site-Directed; Nitrophenols; Protein Engineering; Protein Structure, Tertiary; Threonine; Zinc

This study was designed to determine the concentrations of p-nitrophenyl acetate esterase activity (EA) and cortisol in serum of marine Olive Ridley turtles (Lepidochelys olivacea) from a Mexican Pacific population ("La Escobilla" beach) and to evaluate the possible relationship of inorganic elements with these biomarkers. EA, cortisol, and selected chemical elements (Cd, Pb, Ti, Sr, Se, Al, As, and Zn) were measured in the blood of 44 sea turtles from the Eastern Pacific (Southeast Mexico). Serum EA ranged from 0.4 to 3.9 UI mL

Topics: Animals; Biomarkers; Ecotoxicology; Environmental Monitoring; Environmental Pollutants; Environmental Pollution; Esterases; Female; Hydrocortisone; Metals; Mexico; Nitrophenols; Turtles

Human serum albumin (HSA) has two major ligand-binding sites, sites I and II, and hydrolyzes compounds at both sites. Although the hydrolytic interaction of ester-type drugs with other drugs by HSA has been reported, there are only a few studies concerning the effect of pharmaceutical excipients on the hydrolysis of ester-type drugs by HSA. In the present study, we investigated the effect of ethanol (2 vol%; 345 mM) on the hydrolysis of aspirin, p-nitrophenyl acetate, and olmesartan medoxomil, which are ester-type drugs, with 4 different lots of HSA preparations. The hydrolysis activities of HSA toward aspirin, p-nitrophenyl acetate, and olmesartan medoxomil were measured from the pseudo-first-order degradation rate constant (k

Topics: Anti-Inflammatory Agents, Non-Steroidal; Anticoagulants; Antihypertensive Agents; Aspirin; Binding Sites; Drug Stability; Ethanol; Excipients; Fatty Acids, Nonesterified; Humans; Hydrolysis; Indomethacin; Kinetics; Ligands; Naproxen; Nitrophenols; Olmesartan Medoxomil; Preservatives, Pharmaceutical; Serum Albumin, Human; Warfarin

Lungs are pharmacologically active organs and the pulmonary drug metabolism is of interest for inhaled drugs design. Carboxylesterases (CESs) are enzymes catalyzing the hydrolysis of many structurally different ester, amide and carbamate chemicals, including prodrugs. For the first time, the presence, kinetics, inhibition and inter-individual variations of the major liver CES isozymes (CES1 and CES2) were investigated in cytosol and microsomes of human lungs from 20 individuals using 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD) as substrates the rates of hydrolysis (V

Topics: Carboxylesterase; Carboxylic Ester Hydrolases; Dose-Response Relationship, Drug; Humans; Lung; Male; Microsomes, Liver; Nitrophenols; Umbelliferones

Pharmacokinetics and pharmacodynamics of irinotecan have been reported to be altered in cancer patients with end-stage kidney disease (ESKD). Carboxylesterase (CES) has an important role in metabolism of irinotecan to its active metabolite, SN-38, in human liver. The purpose of the present study was to investigate whether CES activity was altered in ESKD patients.. The present study investigated the effects of uremic serum, uremic toxins, and fatty acids on the hydrolysis of irinotecan and a typical CES substrate, p-nitrophenyl acetate (PNPA), in human liver microsomes. Normal and uremic serum samples were deproteinized by treatment with methanol were used in the present study.. The present study showed that both normal and uremic serum significantly inhibited CES-mediated metabolism of both irinotecan and PNPA. The inhibition by uremic serum was weaker than that by normal serum, suggesting that CES activity may be higher in ESKD patients. Although four uremic toxins did not affect PNPA metabolism, arachidonic acid inhibited it. There was no difference in inhibitory effect of PNPA metabolism between both mixtures of seven fatty acids used at concentrations equivalent to those present in 10% normal or uremic serum. Interestingly, those mixtures had a more pronounced effect than either 10% normal or uremic serum.. The present study showed that the inhibition of CES activity by uremic serum was weaker than that by normal serum, suggesting that an increase in maximum plasma concentration of SN-38 in cancer patients with ESKD can be attributed to an accelerated CES-mediated irinotecan hydrolysis.

Topics: Carboxylesterase; Case-Control Studies; Fatty Acids; Humans; Irinotecan; Kidney Failure, Chronic; Microsomes, Liver; Nitrophenols; Topoisomerase I Inhibitors; Uremia

Corneal esterases are utilized in the activation of topically applied ester prodrugs. Esterases may also be involved in the metabolism of drugs in posterior eye tissues, but their physiological activity is unknown. Furthermore, extrapolation of the esterase activity from protein level to the tissues is missing. The aims of the current study were to determine esterase activities in porcine and albino rabbit ocular tissues, calculate the activities for whole tissues and compare esterase activity between the species. We conducted a hydrolysis study with ocular tissue homogenates using an esterase probe substrate 4-nitrophenyl acetate. The hydrolysis rates were first normalized to protein content and then scaled to whole tissues. The hydrolytic rate normalized to protein content was high in the cornea and iris-ciliary body and low in the lens and aqueous humor, and in general, the rabbit tissues had higher hydrolytic rates than the porcine ones. Esterase activity scaled to whole tissue was high in cornea and iris-ciliary body and low in aqueous humor and retinal pigment epithelium in both species. The current study revealed differences in esterase activities among the ocular tissues and the species. This basic knowledge on ocular esterases provides background information particularly for posterior segment drug development.

Topics: Animals; Enzyme Activation; Esterases; Eye; Female; Hydrolysis; Male; Nitrophenols; Rabbits; Species Specificity; Swine

Esterases catalyze the cleavage and formation of ester bonds and are members of the diverse family of α/β hydrolase fold. They are useful in industries from different sectors, such as food, detergent, fine chemicals, and biofuel production. In a previous work, 30 positive clones for lipolytic activity were identified from a metagenomic library of a microbial consortium specialized in diesel oil degradation. In this study, a putative gene encoding an esterase/lipase, denominated est8, has been cloned and the corresponding protein expressed recombinantly, purified to homogeneity and characterized functional and structurally. We show that the protein codified by est8 gene, denominated Est8, is an alkaline esterase with high catalytic efficiency against p-nitrophenyl acetate and stable in the presence of up to 10% dimethyl sulfoxide. The three-dimensional structure of Est8 was determined at 1.85-Ǻ resolution, allowing the characterization of the substrate-binding pocket and features that rationalize the preference of Est8 for short-chain substrates. In an attempt to increase the size of ligand-binding pocket and enzyme activity against distinct substrates of long chain, we mutated two residues (Met

Topics: Butyrates; Cloning, Molecular; Crystallization; Crystallography, X-Ray; Enzyme Stability; Esterases; Gene Library; Hydrogen-Ion Concentration; Hydrolysis; Lipase; Lipolysis; Metagenomics; Microbial Consortia; Nitrophenols; Recombinant Proteins; Substrate Specificity

An automated spectrophotometric method for total esterase activity (TEA) measurement in porcine saliva has been developed and validated, using 4-nitrophenyl acetate (4-NA) as substrate. The method was precise and accurate, with low limit of detection, and was able to measure samples with TEA activities up to 400IU/L without any dilution. In addition, the different enzymes contributing to TEA were characterized, being identified carbonic anhydrase VI (CA-VI), lipase, cholinesterase (ChE) and cholesterol esterase (CEL). TEA significantly increased (1.49-fold, P<0.01) in healthy pigs just after applying an acute stress stimulus consisting of nasal restraint, being lipase and cholinesterase the main responsible of this increase. TEA was significantly increased (1.83-fold, P<0.001) in a group of pigs with lameness; in this case, in addition to lipase and ChE, CA-VI also increased. The results found in this report indicate that TEA can be easily measured in porcine saliva with an accurate and highly reproducible automated method. Salivary TEA is mainly due to the activity of four enzymes: CA-VI, lipase, ChE and CEL, and these enzymes can change in a different way in situations of stress or disease.

Topics: Animals; Carbonic Anhydrases; Cholinesterases; Esterases; Lipase; Nitrophenols; Reproducibility of Results; Saliva; Spectrophotometry; Sterol Esterase; Swine

Fucoidan is an α-l-fucopyranosyl polymer found in seaweeds with forms that have acetyl and sulfuric modifications and derivatives that are lower and/or diversified, with modifications that have attracted interest as potential bioactive substances. We identified the gene for a fucoidan deacetylase that cleaves acetyl moieties from fucoidan and thereby contributes to fucoidan utilization in the marine bacterium Luteolibacter algae H18. Fucoidan deacetylase was purified to homogeneity from a cell-free extract of L. algae H18, and used to determine the internal amino acid sequence and identify the gene, fud, in a draft genome sequence of the H18 strain. The gene product was heterologously produced in Escherichia coli and was demonstrated to catalyze fucoidan deacetylation, but not desulfation, and degradation into lower forms. In addition to fucoidan deacetylation, the enzyme catalyzed the hydrolysis of p-nitrophenyl esters with organic acids, and p-nitrophenyl acetate was the best substrate among those tested. The present study provides a new tool for fucoidan degradation, potentially expanding investigations on fucoidan derivatives.

Topics: Amino Acid Sequence; Hydrolases; Hydrolysis; Nitrophenols; Phaeophyceae; Polysaccharides; Seaweed

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p-nitrophenyl acetate (pNPA) and 4-nitrophenyl-(2-phenyl)-propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single-histidine residue (His

Topics: Binding Sites; Calcium; Catalysis; Esterases; Histidine; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Metabolic Engineering; Mutagenesis, Site-Directed; Nitrophenols; Nuclear Magnetic Resonance, Biomolecular

Membrane plays significant role in cellular enzymatic reactions. To better understand its function on membrane integral or bound enzymes, DNA origami and frame-guided assembly strategy are combined to construct a given-size, addressable enzyme-containing nanomembrane as a heterogeneous reactor to explore the enzymatic catalyst reaction on the membrane. The enzymes in the membrane are located precisely. This new kind of membrane can enrich hydrophobic substrate molecules in aqueous solution to the embedded enzymes. Otherwise, this nanomembrane shows the capability of substrate selectivity, which plays important role in the highly efficient and specific properties of enzymes in vivo.

Topics: Biocatalysis; DNA; Lipase; Nanostructures; Nitrophenols; Nucleic Acid Conformation

The best-studied amidase signature (AS) enzyme is probably fatty acid amide hydrolase (FAAH). Closely related to FAAH is mandelamide hydrolase (MAH), whose substrate specificity and mechanism of catalysis are described in this paper. First, we developed a convenient chromogenic substrate, 4-nitrophenylacetamide, for MAH. The lack of reactivity of MAH with the corresponding ethyl ester confirmed the very limited size of the MAH leaving group site. The reactivity of MAH with 4-nitrophenyl acetate and methyl 4-nitrophenyl carbonate, therefore, suggested formation of an "inverse" acyl-enzyme where the small acyl-group occupies the normal leaving group site. We have interpreted the specificity of MAH for phenylacetamide substrates and small leaving groups in terms of its active site structure, using a homology model based on a FAAH crystal structure. The relevant structural elements were compared with those of FAAH. Phenylmethylboronic acid is a potent inhibitor of MAH (K

Topics: Amidohydrolases; Bacterial Proteins; Binding Sites; Carbonates; Catalysis; Catalytic Domain; Crystallization; Hydrolases; Hydrolysis; Hydroxamic Acids; Kinetics; Mandelic Acids; Molecular Conformation; Mutagenesis, Site-Directed; Nitrophenols; Pseudomonas putida; Substrate Specificity

Resistance to acaricides in ticks is becoming increasingly widespread throughout the world; therefore, tick control requires resistance monitoring for each tick species. The aims of this study were to monitor the susceptibility status of the cattle tick, Rhipicephalus (Boophilus) annulatus (Acari: Ixodidae), against pyrethroid acaricides from Mazandaran Province, northern Iran, and where resistance was evident, and establish the possible underlying mechanisms. Fully engorged adult R. (B.) annulatus females collected on cattle from Mazandaran Province. Twenty-nine tick populations produced 10-18days old larvae and bioassayed with cypermethrin and λ-cyhalothrin by larval packet test and the levels of detoxification enzymes were measured. Population AM-29 had a maximum resistance ratio (RR

Topics: Acaricides; Animals; Arthropod Proteins; Cytochrome P-450 Enzyme System; Drug Resistance; Female; Glutathione Transferase; Iran; Larva; Nitriles; Nitrophenols; Pyrethrins; Rhipicephalus

Human carboxylesterase (CES) is a key esterase involved in the metabolism and biotransformation of drugs. Hydrolysis activity in the human small intestine is predominantly mediated by CES2A1 rather than CES1A. In drug development studies, Caco-2 cells are commonly used as a model to predict drug absorption in the human small intestine. However, the expression patterns of CES2A1 and CES1A in Caco-2 cells differ from those in the human small intestine. There are also species-specific differences in CES expression patterns between human and experimental animals. Furthermore, it is difficult to obtain primary human intestinal epithelial cells. Therefore, there is currently no system that can precisely predict features of drug absorption, such as CES-mediated metabolism, in the human intestine. To develop a novel system to evaluate intestinal pharmacokinetics, we analyzed CES expression and function in human induced pluripotent stem (iPS) cell-derived enterocytes. CES2A1 mRNA and protein levels in human iPS cell-derived enterocytes were comparable to Caco-2 cells, whereas CES1A levels were lower in human iPS cell-derived enterocytes compared with Caco-2 cells. p-nitrophenyl acetate hydrolysis in human iPS cell-derived enterocytes was significantly inhibited by the CES2A1-specific inhibitor telmisartan. Hydrolysis levels of the CES2A1-specific substrate aspirin were similar in human iPS cell-derived enterocytes and Caco-2 cells, whereas hydrolysis of the CES1A-specific substrate monoethylglycylxylidine was observed in Caco-2 cells but not in human iPS cell-derived enterocytes. These findings demonstrated that the expression and activity of CES isozymes in human iPS cell-derived enterocytes are more similar to the human small intestine compared with Caco-2 cells.

Topics: Benzimidazoles; Benzoates; Blotting, Western; Caco-2 Cells; Carboxylesterase; Cell Differentiation; Cell Line; Enterocytes; Gene Expression; Humans; Hydrolysis; Induced Pluripotent Stem Cells; Intestine, Small; Isoenzymes; Nitrophenols; Reverse Transcriptase Polymerase Chain Reaction; Substrate Specificity; Telmisartan

Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site.

Topics: Butyrates; Catalytic Domain; Clostridium botulinum; Crystallography, X-Ray; Esterases; Hydrolysis; Models, Molecular; Nitrophenols; Polyesters; Protein Conformation; Substrate Specificity; Zinc

Organophosphorus (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxication can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON1) are considered factors underlying age-related sensitivity differences. We used an in vitro system to measure detoxication of AChE-inhibiting pesticides mediated via these esterases. Recombinant human AChE was used as a bioassay of inhibitor concentration following incubation with detoxifying tissue: liver plus Ca(+2) (to stimulate PON1s, measuring activity of both esterases) or EGTA (to inhibit PON1s, thereby measuring CaE activity). AChE inhibitory concentrations of aldicarb, chlorpyrifos oxon, malaoxon, methamidophos, oxamyl, paraoxon, and methylparaoxon were incubated with liver homogenates from adult male rat or one of 20 commercially provided human (11-83 years of age) liver samples. Detoxication was defined as the difference in inhibition produced by the pesticide alone and inhibition measured in combination with liver plus Ca(+2) or liver plus EGTA. Generally, rat liver produced more detoxication than did the human samples. There were large detoxication differences across human samples for some pesticides (especially malaoxon, chlorpyrifos oxon) but not for others (e.g., aldicarb, methamidophos); for the most part these differences did not correlate with age or sex. Chlorpyrifos oxon was fully detoxified only in the presence of Ca(+2) in both rat and human livers. Detoxication of paraoxon and methylparaoxon in rat liver was greater with Ca(+2), but humans showed less differentiation than rats between Ca(+2) and EGTA conditions. This suggests the importance of PON1 detoxication for these three OPs in the rat, but mostly only for chlorpyrifos oxon in human samples. Malaoxon was detoxified similarly with Ca(+2) or EGTA, and the differences across humans correlated with metabolism of p-nitrophenyl acetate, a substrate for CaEs. This suggests the importance of CaEs in malaoxon detoxication. Understanding these individual differences in detoxication can inform human variability in pesticide sensitivity.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Aryldialkylphosphatase; Calcium; Calcium Chelating Agents; Carboxylesterase; Child; Cholinesterase Inhibitors; Egtazic Acid; Female; Humans; Insecticides; Liver; Male; Middle Aged; Nitrophenols; Rats; Rats, Long-Evans; Species Specificity; Young Adult

Two functionalized SBA-15 [amine-functionalized SBA-15 (AFS) and epoxy-functionalized SBA-15 (GFS)] with different types of functional groups were synthesized by a hydrothermal process and post functionalized with 3-aminopropyltriethoxysilane (APTES) and 3-glycidyloxypropyltrimethoxysilane (GPTMS), respectively. They were used for the immobilization of carbonic anhydrase (CA). The physicochemical properties of the functionalized SBA-15 were characterized by X-ray powder diffraction (XRD), N2 adsorption-desorption, (13)C, (29)Si solid-state nuclear magnetic resonance (NMR) spectroscopy, and scanning electron microscopy (SEM). Before and after CA was immobilized on AFS and GFS, the effects of temperature and pH value on the enzyme activity, storage stability, and reusability were investigated using para-nitrophenyl acetate (p-NPA) assay. CA/GFS showed a better performance with respect to storage stability and reusability than CA/AFS. Moreover, the amount of CaCO3 precipitated over CA/AFS was less than that precipitated over CA/GFS, which was almost equal to that precipitated over the free CA. The results indicate that the epoxy group is a more suitable functional group for covalent bonding with CA than the amino group, and GFS is a promising support for CA immobilization.

Topics: Adsorption; Amines; Carbonic Anhydrases; Chemical Precipitation; Drug Storage; Enzyme Stability; Enzymes, Immobilized; Epoxy Compounds; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Nitrophenols; Propylamines; Silanes; Silicon Dioxide; Temperature; X-Ray Diffraction

Human serum albumin (HSA) has two major ligand-binding sites, sites I and II, and also hydrolyzes some compounds at both sites. In the present study, we investigated differences in esterase activity among HSA preparations, and also the effects of warfarin, indomethacin, and naproxen on the hydrolytic activities of HSA to aspirin and p-nitrophenyl acetate. The esterase activities of HSA to aspirin or p-nitrophenyl acetate were measured from the pseudo-first-order formation rate constant (kobs) of salicylic acid or p-nitrophenol by HSA. Inter-lot variations were observed in the esterase activities of HSA to aspirin and p-nitrophenyl acetate; however, the esterase activity of HSA to aspirin did not correlate with that to p-nitrophenyl acetate. The inhibitory effects of warfarin and indomethacin on the esterase activity of HSA to aspirin were stronger than that of naproxen. In contrast, the inhibitory effect of naproxen on the esterase activity of HSA to p-nitrophenyl acetate was stronger than those of warfarin and indomethacin. These results suggest that the administration of different commercial HSA preparations and the co-administration with site I or II high-affinity binding drugs may change the pharmacokinetic profiles of drugs that are hydrolyzed by HSA.

Topics: Aspirin; Esterases; Humans; Hydrolysis; Indomethacin; Naproxen; Nitrophenols; Serum Albumin; Sulfhydryl Compounds; Warfarin

The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.

Topics: Carboxylic Ester Hydrolases; Catalysis; Catalytic Domain; Hydrolysis; Kinetics; Mutation; Nitrophenols; Protein Conformation, alpha-Helical; Protein Engineering; Protein Structure, Tertiary

Mutations the in human DJ-1 (hDJ-1) gene are associated with early-onset autosomal recessive forms of Parkinson's disease (PD). hDJ-1/parkinsonism associated deglycase (PARK7) is a cytoprotective multi-functional protein that contains a conserved cysteine-protease domain. Given that cysteine-proteases can act on both amide and ester substrates, we surmised that hDJ-1 possessed cysteine-mediated esterase activity. To test this hypothesis, hDJ-1 was overexpressed, purified and tested for activity towards 4-nitrophenyl acetate (pNPA) as µmol of pNPA hydrolyzed/min/mg·protein (U/mg protein). hDJ-1 showed maximum reaction velocity esterase activity (Vmax = 235.10 ± 12.00 U/mg protein), with a sigmoidal fit (S0.5 = 0.55 ± 0.040 mM) and apparent positive cooperativity (Hill coefficient of 2.05 ± 0.28). A PD-associated mutant of DJ-1 (M26I) lacked activity. Unlike its protease activity which is inactivated by reactive oxygen species (ROS), esterase activity of hDJ-1 is enhanced upon exposure to low concentrations of hydrogen peroxide (<10 µM) and plateaus at elevated concentrations (>100 µM) suggesting that its activity is resistant to oxidative stress. Esterase activity of DJ-1 requires oxidation of catalytic cysteines, as chemically protecting cysteines blocked its activity whereas an oxido-mimetic mutant of DJ-1 (C106D) exhibited robust esterase activity. Molecular docking studies suggest that C106 and L126 within its catalytic site interact with esterase substrates. Overall, our data show that hDJ-1 contains intrinsic redox-sensitive esterase activity that is abolished in a PD-associated mutant form of the hDJ-1 protein.

Topics: Esterases; Humans; Hydrogen Peroxide; Molecular Docking Simulation; Mutation; Nitrophenols; Oxidation-Reduction; Oxidative Stress; Parkinson Disease; Protein Deglycase DJ-1; Reactive Oxygen Species

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library(1). We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

Topics: Alkaline Phosphatase; Base Sequence; Cellulase; Enzymes; Escherichia coli; Flow Cytometry; Gene Library; Glucosides; High-Throughput Screening Assays; Lipase; Metagenomics; Nitrophenols; Organophosphorus Compounds; Substrate Specificity

Organophosphate insecticides (OPs) continue to be an important class of agrochemicals used in modern agriculture worldwide. Even though these pesticides persist in the environment for a relatively short time, they show a high acute toxicity that may represent a serious hazard for wildlife. Sub-lethal effects on non-target species are a focus in pest management programs and should be used as biomarkers. Cholinesterases (ChEs) are the most used biomarker of OP exposure in vertebrate and invertebrate species. However, the combined monitoring of ChE and carboxylesterase (CE) activities may provide a more useful indication of exposure and effect of the organisms. The objective of the present work was to find the most sensitive combination of enzyme, substrate, tissue and capacity to recovery of B-esterases in the freshwater gastropod Planorbarius corneus exposed to the OP azinphos-methyl. For this purpose, ChE and CE activities in different tissues of P. corneus (head-foot, pulmonary region, digestive gland, gonads and whole organism soft tissue) were studied. Measurements of ChE activity were performed using three substrates: acetylthiocholine, propionylthiocholine and butyrylthiocholine and CE activity using four different substrates: p-nitrophenyl acetate, p-nitrophenyl butyrate, 1-naphthyl acetate, and 2-naphthyl acetate in control and exposed organisms. Finally, the recovery rates of ChE and CE activities following 48h exposure to azinphos-methyl were analyzed. Our results show a preference for acetylthiocholine as substrate, a high inhibition with eserine (a selective ChE inhibitor) and inhibition with excess of substrate in all the analyzed tissues. The highest ChE and CE activity was found in the pulmonary region and in the digestive gland, respectively. The highest CE V

Topics: Animals; Azinphosmethyl; Biomarkers; Butyrates; Carboxylic Ester Hydrolases; Cholinesterases; Inhibitory Concentration 50; Insecticides; Kinetics; Nitrophenols; Organophosphorus Compounds; Snails; Substrate Specificity; Water Pollutants, Chemical

A series of arenesulfonyl-2-imidazolidinones incorporating methyl, isopropyl, methoxy, halogen and phenyl moieties were prepared and tested as possible inhibitors of two members of the pH regulatory enzyme family, carbonic anhydrase (CA; EC 4.2.1.1). The inhibitory potencies of the compounds against human isoforms hCA I and hCA II were analyzed by an esterase assay with 4-nitrophenyl acetate as substrate, and the inhibition constants (KI) were calculated. Most compounds investigated here exhibited micromolar inhibition constants against the two isoenzymes. KI values were in the range of 10.2-40.6 μM for hCA I and of 13.1-31.4 μM for hCA II, respectively. Most of the imidazolidinones showed interesting CA inhibitory efficacy, some of them having comparable affinity (for hCA I) as the clinically used sulfonamide acetazolamide (AZA), but their efficacy against hCA II was much lower compared to AZA.

Topics: Acetazolamide; Carbonic Anhydrase I; Carbonic Anhydrase II; Carbonic Anhydrase Inhibitors; Enzyme Assays; Humans; Imidazolidines; Kinetics; Nitrophenols; Recombinant Proteins; Structure-Activity Relationship

A new series of chiral thiourea derivatives (5a-5c) and thiourea containing benzimidazole moieties (9b-9e) were synthesized from different amino acids (l-valine, l-isoleucine, l-methionine, l-phenylalanine, and d-phenylglycine). The compounds were characterized and tested against the two most studied members of the pH regulatory enzyme family, carbonic anhydrase (CA, EC 4.2.1.1). KI values of the novel compounds were measured in the range of 3.4-73.6 μM for hCA I isozyme and 8.7-1.44.2 μM for hCA II isozyme, respectively. Phenol was also tested as standard in order to understand the structure activity relationship and the clinically used sulfonamide acetazolamide was tested for comparison reasons. All of the compounds exhibited competitive inhibition with 4-nitrophenylacetate as substrate.

Topics: Acetazolamide; Amino Acids; Benzimidazoles; Carbonic Anhydrase I; Carbonic Anhydrase II; Carbonic Anhydrase Inhibitors; Enzyme Assays; Humans; Kinetics; Nitrophenols; Phenol; Recombinant Proteins; Structure-Activity Relationship; Thiourea

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

Topics: Bioengineering; Carboxylic Ester Hydrolases; Crystallization; Directed Molecular Evolution; Kinetics; Models, Molecular; Nitrophenols; Protein Conformation; Protein Folding; Small Molecule Libraries; Substrate Specificity

A system performing both a catalytic hydrolysis reaction and the direct optical monitoring of the product was created by the combination of two bolaamphiphile self-assemblies. Two bolaamphiphilic self-assemblies were applied as a biomimetic catalyst of p-nitrophenyl acetate (p-NPA) hydrolysis and an optical sensor probe that detects p-NPA hydrolysis through photoluminescence quenching by p-nitrophenol (p-NP), the product of p-NPA hydrolysis. One bolaamphiphilic self-assembly with a histidine moiety catalytically hydrolyzed the p-NPA substrate, and the other self-assembly of tyrosyl bolaamphiphile monitored the product of p-NP by photoluminescence quenching. The progression of the reaction and quenching degree were adjusted by controlling the quantity of histidyl and tyrosyl self-assemblies, respectively. The reaction and subsequent sensing cascade could be interrupted by a reducing agent. The addition of NaBH4 induced the chemical conversion of p-NP to p-aminophenol, which retarded photoluminescence quenching. Thus, it was demonstrated that hydrolysis of an organic substrate and subsequent monitoring of the hydrolysis reaction could be achieved through a combination of independent bolaamphiphilic self-assemblies. This study demonstrated the construction of a catalytic reaction and detection system incorporating designer biomimetic self-assemblies whose functionalities were devised to realize deliberate functions.

Topics: Biomimetic Materials; Biosensing Techniques; Catalysis; Furans; Histidine; Hydrolysis; Nitrophenols; Pyridones; Spectrometry, Fluorescence; Tyrosine

In this study we use a straightforward experimental method to probe the presence and activity of the proteolytic enzyme α-chymotrypsin adsorbed on titania colloidal particles. We show that the adsorption of α-chymotrypsin on the particles is irreversible and pH-dependent. At pH 8 the amount of adsorbed chymotrypsin is threefold higher compared to the adsorption at pH 5. However, we observe that the adsorption is accompanied by a substantial loss of enzymatic activity, and only around 6-9% of the initial enzyme activity is retained. A Michaelis-Menten kinetics analysis of both unbound and TiO2-bound chymotrypsin shows that the K(M) value is increased from ∼10 μM for free chymotrypsin to ∼40 μM for the particle bound enzyme. Such activity decrease could be related by the hindered accessibility of substrate to the active site of adsorbed chymotrypsin, or by adsorption-induced structural changes. Our simple experimental method does not require any complex technical equipment, can be applied to a broad range of hydrolytic enzymes and to various types of colloidal materials. Our approach allows an easy, fast and reliable determination of particle surface-bound enzyme activity and has high potential for development of future enzyme-based biotechnological and industrial processes.

Topics: Adsorption; Catalytic Domain; Chymotrypsin; Colloids; Enzyme Assays; Enzymes, Immobilized; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Kinetics; Nitrophenols; Titanium

Carboxylesterase 1 (CES1) is the major hydrolase in human liver. The enzyme is involved in the metabolism of several important therapeutic agents, drugs of abuse, and endogenous compounds. However, no studies have described the role of human CES1 in the activation of two commonly prescribed angiotensin-converting enzyme inhibitors: enalapril and ramipril. Here, we studied recombinant human CES1- and CES2-mediated hydrolytic activation of the prodrug esters enalapril and ramipril, compared with the activation of the known substrate trandolapril. Enalapril, ramipril, and trandolapril were readily hydrolyzed by CES1, but not by CES2. Ramipril and trandolapril exhibited Michaelis-Menten kinetics, while enalapril demonstrated substrate inhibition kinetics. Intrinsic clearances were 1.061, 0.360, and 0.02 ml/min/mg protein for ramipril, trandolapril, and enalapril, respectively. Additionally, we screened a panel of therapeutic drugs and drugs of abuse to assess their inhibition of the hydrolysis of p-nitrophenyl acetate by recombinant CES1 and human liver microsomes. The screening assay confirmed several known inhibitors of CES1 and identified two previously unreported inhibitors: the dihydropyridine calcium antagonist, isradipine, and the immunosuppressive agent, tacrolimus. CES1 plays a role in the metabolism of several drugs used in the treatment of common conditions, including hypertension, congestive heart failure, and diabetes mellitus; thus, there is a potential for clinically relevant drug-drug interactions. The findings in the present study may contribute to the prediction of such interactions in humans, thus opening up possibilities for safer drug treatments.

Topics: Angiotensin-Converting Enzyme Inhibitors; Carboxylesterase; Carboxylic Ester Hydrolases; Diltiazem; Drug Interactions; Enalapril; Esters; Humans; Hydrolysis; Inactivation, Metabolic; Indoles; Kinetics; Liver; Microsomes, Liver; Nitrophenols; Prodrugs; Ramipril; Recombinant Proteins; Verapamil

Recent advances in protein design have opened avenues for the creation of artificial enzymes needed for biotechnological and pharmaceutical applications. However, designing efficient enzymes remains an unrealized ambition, as the design must incorporate a catalytic apparatus specific for the desired reaction. Here we present a de novo design approach to evolve a minimal carbonic anhydrase mimic. We followed a step-by-step design of first folding the main chain followed by sequence variation for substrate binding and catalysis. To optimize the fold, we designed an αββ protein based on a Zn-finger. We then inverse-designed the sequences to provide stability to the fold along with flexibility of linker regions to optimize Zn binding and substrate hydrolysis. The resultant peptides were synthesized and assessed for Zn and substrate binding affinity by fluorescence and ITC followed by evaluation of catalytic efficiency with UV-based enzyme kinetic assays. We were successful in mimicking carbonic anhydrase activity in a peptide of twenty two residues, using p-nitrophenyl acetate as a CO2 surrogate. Although our design had modest activity, being a simple structure is an advantage for further improvement in efficiency. Our approach opens a way forward to evolving an efficient biocatalyst for any industrial reaction of interest.

Topics: Amino Acid Sequence; Biocatalysis; Biomimetics; Carbonic Anhydrases; Computational Biology; Hydrolases; Hydrolysis; Kinetics; Models, Molecular; Molecular Sequence Data; Nitrophenols; Organophosphorus Compounds; Peptides; Protein Binding; Protein Engineering; Protein Folding; Protein Structure, Secondary; Proton Magnetic Resonance Spectroscopy; Spectrometry, Fluorescence; Substrate Specificity; Zinc; Zinc Fingers

It is shown by rational site-directed mutagenesis of the lid region in Thermomyces lanuginosus lipase that it is possible to generate lipase variants with attractive features, e.g., high lipase activity, fast activation at the lipid interface, ability to act on water-soluble substrates, and enhanced calcium independence. The rational design was based on the lid residue composition in Aspergillus niger ferulic acid esterase (FAEA). Five constructs included lipase variants containing the full FAEA lid, a FAEA-like lid, an intermediate lid of FAEA and TlL character, and the entire lid region from Aspergillus terreus lipase (AtL). To investigate an altered activation mechanism for each variant compared to that of TlL, a combination of activity- and spectroscopic-based measurements were applied. The engineered variant with a lid from AtL displayed interfacial activation comparable to that of TlL, whereas variants with FAEA lid character showed interfacial activation independence with pronounced activity toward pNP-acetate and pNP-butyrate below the critical micelle concentration. For variants with lipase and esterase character, lipase activity measurements further indicated a faster activation at the lipid interface. Relative to their activity toward pNP-ester substrates in calcium-rich buffer, all lid variants retained between 15 and 100% activity in buffer containing 5 mM EDTA whereas TlL activity was reduced to less than 2%, demonstrating the lid's central role in governing calcium dependency. For FAEA-like lid variants, accessible hydrophobic surface area measurements showed an approximate 10-fold increase in the level of binding of extrinsic fluorophores to the protein surface relative to that of TlL accompanied by a blue shift in emission indicative of an open lid in aqueous solution. Together, these studies report on the successful alteration of the activation mechanism in TlL by rational design creating novel lipases with new, intriguing functionalities.

Topics: Amino Acid Sequence; Aspergillus; Butyrates; Carboxylic Ester Hydrolases; Decanoates; Enzyme Activation; Eurotiales; Fungal Proteins; Hydrolysis; Hydrophobic and Hydrophilic Interactions; Lipase; Models, Molecular; Molecular Sequence Data; Mutation; Nitrophenols; Protein Conformation

Electrospun membranes were studied for the chemical deactivation of threat agents by means of enzymatic proteins. Protein loading and the surface chemistry of hybrid nanofibers influenced the efficacy by which embedded enzymes could digest the substrate of interest. Bovine serum albumin (BSA), selected as a model protein, was electrospun into biologically active fibers of poly(vinyl alcohol), PVA. Single-walled carbon nanotubes (SWNTs) were blended within these mixtures to promote protein assembly during the process of electrospinning and subsequently the ester hydrolysis of the substrates. The SWNT incorporation was shown to influence the topography of PVA/BSA nanofibers and enzymatic activity against paraoxon, a simulant for organophosphate agents and a phosphorus analogue of p-nitrophenyl acetate (PNA). The esterase activity of BSA against PNA was uncompromised upon its inclusion within nanofibrous membranes because similar amounts of PNA were hydrolyzed by BSA in solution and the electrospun BSA. However, the availability of BSA along the fiber surface was shown to affect the ester hydrolysis of paraoxon. Atomic force microscopy images of nanofibers implicated the surface migration of BSA during the electrospinning of SWNT filled dispersions, especially as greater weight fractions of protein were added to the spinning mixtures. In turn, the PVA/SWNT/BSA nanofibers outperformed the nanotube free PVA/BSA membranes in terms of paraoxon digestion. The results support the development of electrospun polymer nanofiber platforms, modulated by SWNTs for enzyme catalytic applications relevant to soldier protective ensembles.

Topics: Animals; Cattle; Esters; Hydrolysis; Membranes, Artificial; Nanotubes, Carbon; Nitrophenols; Polyvinyl Alcohol; Serum Albumin, Bovine

Mycobacterium tuberculosis possesses an unusually high number of genes involved in the metabolism of lipids. Driven by a newly described esterase motif SXXK in the amino acid sequence and a predicted signal peptide, the gene rv3036c from M. tuberculosis was cloned and characterized biochemically. Rv3036c efficiently hydrolyzes soluble p-nitrophenyl esters but not emulsified lipid. The highest activity of this enzyme was observed when p-nitrophenyl acetate (C2) was used as the substrate. Based on the activities, Rv3036c was classified as a nonlipolytic hydrolase. The results of immunoreactivity studies on the subcellular mycobacterial fractions suggested that the enzyme was present in the cell wall and cell membrane in mycobacteria. In summary, Rv3036c was characterized as a novel cell wall-anchored esterase from M. tuberculosis.

Topics: Amino Acid Sequence; Enzyme Activation; Escherichia coli; Esterases; Mycobacterium tuberculosis; Nitrophenols; Protein Sorting Signals

Carbonic anhydrase enzymes catalyse the reversible hydration of carbon dioxide to bicarbonate. A thermophilic Thermovibrio ammonificans α-carbonic anhydrase (TaCA) has been expressed in Escherichia coli and structurally and biochemically characterized. The crystal structure of TaCA has been determined in its native form and in two complexes with bound inhibitors. The tetrameric enzyme is stabilized by a unique core in the centre of the molecule formed by two intersubunit disulfides and a single lysine residue from each monomer that is involved in intersubunit ionic interactions. The structure of this core protects the intersubunit disulfides from reduction, whereas the conserved intrasubunit disulfides are not formed in the reducing environment of the E. coli host cytosol. When oxidized to mimic the environment of the periplasmic space, TaCA has increased thermostability, retaining 90% activity after incubation at 70°C for 1 h, making it a good candidate for industrial carbon-dioxide capture. The reduction of all TaCA cysteines resulted in dissociation of the tetrameric molecule into monomers with lower activity and reduced thermostability. Unlike other characterized α-carbonic anhydrases, TaCA does not display esterase activity towards p-nitrophenyl acetate, which appears to result from the increased rigidity of its protein scaffold.

Topics: Acetazolamide; Bacteria; Bacterial Proteins; Carbon Dioxide; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Catalytic Domain; Cloning, Molecular; Crystallography, X-Ray; Disulfides; Enzyme Stability; Kinetics; Models, Molecular; Nitrophenols; Protein Conformation; Sulfanilamide; Sulfanilamides; Temperature

A novel esterase gene was isolated by functional screening of a metagenomic library prepared from an activated sludge sample. The gene (est-XG2) consists of 1,506 bp with GC content of 74.8 %, and encodes a protein of 501 amino acids with a molecular mass of 53 kDa. Sequence alignment revealed that Est-XG2 shows a maximum amino acid identity (47 %) with the carboxylesterase from Thermaerobacter marianensis DSM 12885 (YP_004101478). The catalytic triad of Est-XG2 was predicted to be Ser₁₉₂-Glu₃₁₃-His₄₁₂ with Ser₉₂ in a conserved pentapeptide (GXSXG), and further confirmed by site-directed mutagenesis. Phylogenetic analysis suggested Est-XG2 belongs to the bacterial lipase/esterase family VII. The recombinant Est-XG2, expressed and purified from Escherichia coli, preferred to hydrolyze short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate (K(m) and k(cat) of 0.33 mM and 36.21 s⁻¹, respectively). The purified enzyme also had the ability to cleave sterically hindered esters of tertiary alcohols. Biochemical characterization of Est-XG2 revealed that it is a thermophilic esterase that exhibits optimum activity at pH 8.5 and 70 °C. Est-XG2 had moderate tolerance to organic solvents and surfactants. The unique properties of Est-XG2, high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications.

Topics: Amino Acid Sequence; Biotechnology; Enzyme Stability; Escherichia coli; Esterases; Gene Library; Hydrogen-Ion Concentration; Kinetics; Lipase; Metagenomics; Models, Molecular; Molecular Sequence Data; Molecular Weight; Nitrophenols; Phylogeny; Recombinant Proteins; Sequence Alignment; Sequence Analysis, DNA; Sewage; Substrate Specificity; Temperature

Acetyl xylan esterase (AXE) from basidiomycete Coprinopsis cinerea Okayama 7 (#130) was functionally expressed in Pichia pastoris with a C-terminal tag under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 1.5 mg l(-1). Its molecular mass was estimated to be 65.5 kDa based on the SDS-PAGE analysis, which is higher than the calculated molecular mass of 40 kDa based on amino acid composition. In-silico analysis of the amino acid sequence predicted two potential N-glycosylation sites. Results from PNGase F deglycosylation and mass spectrum confirmed the presence of N-glycosylation on the recombinant AXE with predominant N-glycans HexNAc2Hex9-16. The recombinant AXE showed best activity at 40 °C and pH 8. It showed not only acetyl esterase activity with a Km of 4.3 mM and a Vmax of 2.15 U mg(-1) for hydrolysis of 4-nitrophenyl acetate but also a butyl esterase activity for hydrolysis of 4-nitrophenyl butyrate with a Km of 0.11 mM and Vmax of 0.78 U mg(-1). The presence of two additional amino acid residues at its native N-terminus was found to help stabilize the enzyme against the protease cleavages without affecting its activity.

Topics: Acetylesterase; Agaricales; Butyrates; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Gene Expression; Glycosylation; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Nitrophenols; Pichia; Protein Processing, Post-Translational; Temperature

Carboxylesterases hydrolyze esters, amides, and thioesters to produce carboxylic acids and resulting alcohols, amines, and thiols, respectively. Uridine 5'-diphosphate- glucuronosyltransferases are colocalized with carboxylesterases and have the potential to further metabolize carboxylic acids to acyl glucuronides, but it is currently unknown if acyl glucuronides, being esters, also interact with carboxylesterases.. This study explores the ability of acyl glucuronides to act as substrates or inhibitors of human carboxylesterases 1 (hCES1) and 2 (hCES2).. The stability of six acyl glucuronides in the presence of hCES1, hCES2, and buffer alone (100 mM potassium phosphate, pH 7.4, 37°C) were investigated. Reversible inhibition of 4-nitrophenyl acetate hydrolysis by the acyl glucuronides was also studied. Diclofenac-β-d-glucuronide was used to explore potential time-dependent inactivation.. The chemical stability half-life values for CGP 47292-β-d-glucuronide, diclofenac-β-d-glucuronide, (R)-naproxen-β-d-glucuronide, (S)-naproxen-β-d-glucuronide, ibuprofen-β-d-glucuronide (racemic), clopidogrel-β-d-glucuronide, and valproate-β-d-glucuronide were found to be 0.252, 0.537, 0.996, 1.77, 3.67, 5.02, and 15.2 hours, respectively. Diclofenac-β-d-glucuronide, clopidogrel-β-d-glucuronide, ibuprofen-β-d-glucuronide, (R)-naproxen-β-d-glucuronide, and (S)-naproxen-β-d-glucuronide selectively inhibited hCES1, with Ki values of 4.32 ± 0.47, 24.8 ± 4.2, 355 ± 38, 468 ± 21, 707 ± 64 µM, respectively, but did not significantly inhibit hCES2. Valproate-β-d-glucuronide and CGP 47292-β-d-glucuronide did not inhibit either hCES. Time-dependent inactivation of hCES1 by diclofenac-β-d-glucuronide was not observed. Lastly, both hCES1 and hCES2 were shown not to catalyze the hydrolysis of the acyl glucuronides studied.. Drug-drug interaction studies may be warranted for drugs that metabolize to acyl glucuronides due to the potential inhibition of hCESs.

Topics: Carboxylic Ester Hydrolases; Drug Stability; Enzyme Inhibitors; Glucuronides; Half-Life; Humans; In Vitro Techniques; Isoenzymes; Nitrophenols

Sulfamides represent an important class of biologically active compounds. A series of novel sulfamides were synthesized from 1-aminoindanes, 1-aminotetralin, 2-aminoindanes and 2-aminotetralin via the reactions of free amines, benzyl alcohol and chlorosulfonyl isocyanate (CSI) followed by hydrogenolysis of the obtained sulfamoylcarbamates. Carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects of the new sulfamides have been investigated. The human (h) isozymes hCA I and hCA II have been investigated in this study by using an esterase assay with 4-nitrophenyl acetate as substrate. The new sulfamides showed inhibition constants in the micro-submicromolar range, with one compound (N-(indane-1-yl)sulfamide) showing a Ki of 0.45μM against hCA I and of 1.07μM against hCA II.

Topics: Carbonic Anhydrase I; Carbonic Anhydrase II; Carbonic Anhydrase Inhibitors; Erythrocytes; Humans; Kinetics; Nitrophenols; Protein Binding; Structure-Activity Relationship; Substrate Specificity; Sulfonamides

Topics: Catalysis; Decarboxylation; Indole Alkaloids; Nitrophenols; Palladium; Potassium Acetate

A novel oligomeric hydrolase (LI22) from Listeria innocua CLIP 11262 was identified, characterized, and immobilized for industrial application. Sequence analysis of LI22 revealed a putative catalytic triad (Ser(10)-Asp(176)-His(179)), and a conserved sequence motif Ser(S)(10)-Gly(G)(77)-Asn(N)(79)-His(H)(179) with moderate identities (<30%) with other members of the SGNH-hydrolase superfamily. LI22 was able to hydrolyze p-nitrophenyl acetate, α- and β-naphthyl acetate, while the S10A mutant completely lost its activity. Structural properties of LI22 were investigated using gel filtration, circular dichroism (CD), fluorescence, molecular modeling, and gel filtration. We have shown that upon incubation in 30% TFE or 50% ethanol solution, LI22 was transformed into curly amyloid fibrils. Cross-linked enzyme aggregates of LI22 were prepared by precipitating the enzyme with ammonium sulfate and subsequent cross-linking with glutaraldehyde. Higher thermal and chemical stability, as well as good durability after repeated use of the LI22-CLEA, highlight its potential applicability as a biocatalyst in the pharmaceutical and chemical industries.

Topics: Amino Acid Sequence; Amyloid; Benzothiazoles; Carboxylic Ester Hydrolases; Catalysis; Chromatography, Gel; Circular Dichroism; Cross-Linking Reagents; Enzymes, Immobilized; Escherichia coli; Hydrolysis; Listeria; Models, Molecular; Molecular Sequence Data; Mutation; Nitrophenols; Sequence Homology, Amino Acid; Spectrometry, Fluorescence; Thiazoles

To evaluate and validate 3 spectrophotometric assays for measuring serum activity of paraoxonase type-1 (PON1), an enzyme associated with high-density lipoproteins, in dogs.. 22 healthy adult dogs and 10 dogs with eccentrocytosis.. 2 methods were adapted for use in 96-well microplates with phenyl acetate and 5-thiobutyl butyrolactonase as substrates, and 1 was adapted for use in an automated analyzer with p-nitrophenyl acetate as substrate. Blood samples were collected from all dogs, serum was harvested, and serum PON1 activity was measured with each method.. Imprecision was low for all 3 methods, with the exception of interassay imprecision for 5-thiobutyl butyrolactonase, and results were linear across serial sample dilutions. The 3 methods were able to detect low PON1 activity when EDTA was used for blood sample collection, yielded lower PON1 values in sick dogs with eccentrocytosis than in healthy dogs, and yielded highly correlated results.. The methods described here may allow a wider use of PON1 activity as a biomarker of oxidative stress in dogs in clinical and research settings. Results of each method were robust and precise (with the exception of the interassay values for the lactonase method), and the methods were easy to set up in a laboratory.

Topics: Acetates; Animals; Aryldialkylphosphatase; Biomarkers; Carboxylic Ester Hydrolases; Dogs; Female; Male; Nitrophenols; Oxidative Stress; Phenols; Spectrophotometry

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.

Topics: Amino Acid Sequence; Catalysis; Catalytic Domain; Evolution, Molecular; Hydrogen-Ion Concentration; Kinetics; Metalloproteins; Molecular Sequence Data; Nitrophenols; Organophosphorus Compounds; Protein Engineering; Zinc

Immobilization of enzymes usually improves the recyclability and stability and can sometimes also improve the activity compared to enzymes free in solution. Mesoporous silica is a widely studied material as host for immobilized enzymes because of its large internal surface area and tunable pores. It has previously been shown that the pore size is critical both for the loading capacity and for the enzymatic activity; however, less focus has been given to the influence of the particle size. In this work the effect of particle size and particle morphology on the immobilization of lipase from Mucor miehei and Rhizopus oryzae have been investigated. Three kinds of mesoporous silica, all with 9 nm pores but with varying particle size (1000 nm, 300 nm and 40 nm) have been synthesized and were used as host for the lipases. The two lipases, which have the same molecular size but widely different isoelectric points, were immobilized into the silica particles at varied pH values within the interval 5-8. The 300 nm particles were proven to be the most suitable carrier with respect to specific activity for both enzymes. The lipase from M. miehei was more than four times as active when immobilized at pH 8 compared to free in solution whereas the difference was less pronounced for the R. oryzae lipase.

Topics: Adsorption; Enzyme Stability; Enzymes, Immobilized; Fungal Proteins; Hydrogen-Ion Concentration; Isoelectric Point; Lipase; Microscopy, Electron, Scanning; Molecular Weight; Mucor; Nitrophenols; Particle Size; Porosity; Rhizopus; Scattering, Small Angle; Silicon Dioxide; Temperature; X-Ray Diffraction

Human serum albumin (HSA) displays esterase activity reflecting multiple irreversible chemical modifications rather than turnover. Here, kinetics of the pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate (NphOAc) are reported. Under conditions where [HSA]≥ 5×[NphOAc] and [NphOAc]≥ 5×[HSA], the HSA-catalyzed hydrolysis of NphOAc is a first-order process for more than 95% of its course. From the dependence of the apparent rate constants k(app) and k(obs) on [HSA] and [NphOAc], respectively, values of K(s), k(+2), and k(+2)/K(s) were determined. Values of K(s), k(+2), and k(+2)/K(s) obtained at [HSA]≥ 5×[NphOAc] and [NphOAc]≥ 5×[HSA] are in good agreement, the deacylation step being rate limiting in catalysis. The pH-dependence of k(+2)/K(s), k(+2), and K(s) reflects the acidic pK(a) shift of the Tyr411 catalytic residue from 9.0 ± 0.1 in the substrate-free HSA to 8.1 ± 0.1 in the HSA:NphOAc complex. Accordingly, diazepam inhibits competitively the HSA-catalyzed hydrolysis of NphOAc by binding to Tyr411.

Topics: Catalysis; Esterases; Humans; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Nitrophenols; Serum Albumin; Tyrosine

A new carbonic anhydrase (CA, EC 4.2.1.1) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 was identified and characterized. The bacterial carbonic anhydrase gene was expressed in Escherichia coli yielding an active enzyme, which was purified in large amounts. The recombinant protein (SspCA) was found to belong to the α-CA class and displays esterase activity. The kinetic parameters were determined by using CO(2) and p-nitrophenylacetate (p-NpA) as substrates. The bacterial enzyme presented specific activity comparable to that of bovine carbonic anhydrase (bCA II) but it showed biochemical properties never observed for the mammalian enzyme. The thermophilic enzyme, in fact, was endowed with high thermostability and with unaltered residual activity after prolonged exposure to heat up to 100°C. SspCA and the bovine carbonic anhydrase (bCA II) were immobilized within a polyurethane (PU) foam. The immobilized bacterial enzyme was found to be active and stable at 100°C up to 50 h.

Topics: Animals; Bacterial Proteins; Carbon Dioxide; Carbonic Anhydrase II; Carbonic Anhydrases; Cattle; Electrophoresis, Polyacrylamide Gel; Enzyme Assays; Enzyme Stability; Escherichia coli; Gram-Negative Chemolithotrophic Bacteria; Hot Temperature; Immobilized Proteins; Kinetics; Nitrophenols; Polyurethanes; Recombinant Proteins; Substrate Specificity

Topics: Biocatalysis; Chymotrypsin; Deuterium Exchange Measurement; Hydrolysis; Molecular Conformation; Nitrophenols; Nuclear Magnetic Resonance, Biomolecular; Spectrometry, Mass, Electrospray Ionization; Thermodynamics; Time Factors

The mechanism of p-nitrophenyl acetate (pNPA) hydrolysis promoted by vanadate ions was investigated utilizing both density functional theory and ab initio methods. In accordance with experiments, suggesting pure hydrolytic ester bond cleavage involving a nucleophilic addition in the rate-limiting transition state, four possible B(AC)2 (acyl-oxygen bond cleavage) mode reaction pathways were modeled. Moreover, two alternative reaction modes were also considered. Geometry optimizations were carried out using B3LYP, BP86, and MPWB1K functionals, conjugated with a 6-31++G(d,p) basis set and a Stuttgart effective core potential (ECP) for the vanadium atom. Single-point calculations were performed utilizing M06, B3LYP-D, and BP86-D functionals as well as B2PLYP-D and MP2 methods with a 6-311++G(2d,2p) basis set (with and without ECP). To address bulk solvation effects, the universal solvation model (SMD) and the conductor-like polarizable continuum model were applied, using the parameters of water. All levels of theory predict the same reaction mechanism, B(AC)2-1, as the lowest-energy pathway on the potential energy surface for pNPA hydrolysis catalyzed by the H(2)VO(4)(-) ion in aqueous media. The B(AC)2-1 pathway passes through two transition states, the first associated with the nucleophilic addition of H(2)VO(4)(-) and the second with the release of p-nitrophenoxide ion (pNP(-)), linked with a tetrahedral intermediate state. The intermediate structure is stabilized via protonation of the acyl oxygen atom by the vanadate and formation of an intramolecular hydrogen bond. The first and second barrier heights are 24.9 and 1.3 kcal/mol respectively, as calculated with the SMD-M06 approach. The theoretically predicted B(AC)2-1 mechanism is in good agreement with the experiment.

Topics: Anions; Catalysis; Hydrolysis; Molecular Structure; Nitrophenols; Quantum Theory; Vanadates

Hydrolysis of carboxylic esters p-nitrophenyl acetate (pNPA), p-nitrophenyl butyrate (pNPB) and p-nitrophenyl trimethyl acetate (pNPTA) was examined in oxovanadate solutions by means of (1)H and (51)V NMR spectroscopy. In the presence of a mixture of oxovanadates, the hydrolysis of carboxyester bonds in pNPA proceeds under physiological conditions (37 °C, pD = 7.4) with a rate constant of k(obs) = 3.0 × 10(-5) s(-1) representing an acceleration of at least one order of magnitude compared to the uncatalyzed cleavage. EPR and NMR spectra did not show evidence for the formation of paramagnetic species, excluding the possibility of V(+5) reduction to V(+4), and indicating that the cleavage of the carboxyester bond is purely hydrolytic. The pH dependence of k(obs) revealed that the hydrolysis is slow in acidic media but rapidly accelerates in basic solutions. Comparison of the rate profile with the concentration profile of polyoxovanadates shows a clear overlap of the k(obs) profile with the concentration of monovanadate (V(1)). Kinetic experiments at 37 °C using a fixed amount of pNPA and increasing amounts of V(1) permitted the calculation of catalytic (k(c) = 1 x10(-4) s(-1)) and formation constant for the pNPA-V(1) complex (K(f) = 17.5 M(-1)). The (51)V NMR spectra of a reaction mixture revealed broadening and shifting of the (51)V NMR resonances of the V(1) and V(2) upon addition of increasing amount of pNPA, suggesting a dynamic exchange process between vanadates and pNPA, occurring via a rapid association-dissociation equilibrium. The origin of the hydrolytic activity of vanadate is most likely a combination of its nucleophilic nature and the chelating properties which can lead to the stabilization of the transition state.

Topics: Butyrates; Catalysis; Electron Spin Resonance Spectroscopy; Hydrolysis; Kinetics; Magnetic Resonance Spectroscopy; Nitrophenols; Thermodynamics; Vanadates

A thermophilic microorganism, SBS-4S, was isolated from a hot spring located in Gilgit, Northern Areas of Pakistan. It was found to be an aerobic, gram-positive, rod-shaped, thermophilic bacterium that grew on various sugars, carboxylic acids and hydrocarbons at temperatures between 45°C and 75°C. Complete 16S rRNA gene sequence of the microorganism exhibited homology to various species of genus Geobacillus. A highest homology of 99.8% was found with Geobacillus kaustophilus. A partial (0.7 kbp) chaperonin gene sequence also showed a highest homology of 99.4% to that of G. kaustophilus whereas biochemical characteristics of the microorganism were similar to Geobacillus uzenensis. Based on biochemical characterization, 16S rRNA and chaperonin gene sequences, we identified SBS-4S as a strain of genus Geobacillus. Strain SBS-4S produced several extracellular enzymes including amylase, protease and lipase. The lipase encoding gene was cloned, expressed in Escherichia coli and the gene product was characterized. The recombinant lipase was optimally active at 60°C with stability at wide pH range (6-12). The enzyme activity was enhanced remarkably in the presence of Ca(+2). The K(m) and the V(max) for the hydrolysis of p-nitrophenyl acetate were 3.8mM and 2273 μmol min(-1)mg(-1), respectively. The ability of the recombinant enzyme to be stable at a wide pH range makes it a potential candidate for use in industry.

Topics: Amino Acid Sequence; Bacterial Proteins; Cloning, Molecular; Culture Media; DNA, Bacterial; Enzyme Stability; Escherichia coli; Geobacillus; Hot Springs; Hot Temperature; Hydrogen-Ion Concentration; Lipase; Molecular Sequence Data; Molecular Weight; Nitrophenols; Pakistan; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA

Ester precursors of fluorogenic or chromogenic probes are often employed in studies of yeast cell biology. This study was aimed at a comparison of the ability of several commonly used laboratory wild-type Saccharomyces cerevisiae strains to hydrolyse the following model esters: fluorescein diacetate, 2-naphthyl acetate, PNPA (p-nitrophenyl acetate) and AMQI (7-acetoxy-1-methylquinolinum iodide). In all the strains, the esterase activity was localized mainly to the cytosol. Considerable differences in esterase activity were observed between various wild-type laboratory yeast strains. The phase of growth also contributed to the variation in esterase activity of the yeast. This diversity implies the need for caution in using intracellularly hydrolysed probes for a comparison of yeast strains with various genetic backgrounds.

Topics: Cell Size; Cytosol; Esterases; Esters; Fluoresceins; Hydrolysis; Naphthaleneacetic Acids; Nitrophenols; Saccharomyces cerevisiae; Species Specificity; Substrate Specificity

1,2,3-Triazolo[4,5-b]pyridines and pyrazolo[4,3-b]pyridines can be readily prepared via cyanoacetylation reactions of 5-amino-1,2,3-triazoles 1a,b and 4-amino- pyrazole 2 followed by subsequent cyclization of the formed cyanoacetamides. Reactions of amines 1a,b with a mixture of p-nitrophenylacetic acid and acetic anhydride under microwave irradiation conditions afforded the corresponding amides 15a,b that underwent cyclization to form 1,2,3-triazolo[4,5-b]pyridines 16a,b upon heating in DMF solutions containing sodium acetate. Reactions of 1a,b with active methylene compounds, including 17a-c, in the presence of zeolites as catalyst also afforded 1,2,3-triazolo[4,5-b]pyridine derivatives 20a-f via the intermediacy of triazole derivatives 19 and not 18.

Topics: Acetates; Acylation; Amines; Molecular Structure; Nitriles; Nitrophenols; Pyridines; Triazoles; Zeolites

A novel lipase gene from Aspergillus fumigatus, afl1-1, was cloned and expressed with a molecular mass of 38 kDa in Escherichia coli for the first time. The recombinant lipase had a preference for short carbon chain p-nitrophenyl esters, especially toward C2 p-nitrophenyl ester and exhibited potent hydrolysis activity that had not been observed. The optimum pH and temperature of this new enzyme were 8.5 and 65 °C, respectively. The recombinant lipase (AFL1-1) is an alkaline enzyme which was stable in the pH range 6.0∼8.5 for 16 h (at 4 °C) and at 30∼50 °C for 1 h. It is an intracellular enzyme which was purified approximately 8.47-fold with an overall yield of 86.1% by single-step Ni-NTA affinity purification, with a very high specific activity of approximately 1.00 × 10(3) U mg(-1) on a standard substrate of p-nitrophenyl acetate. The Michaelis-Menten kinetic parameters V (max) and K (m) of the lipase were 1.37 mM mg(-1) min(-1) and 14.0 mM, respectively. Ca(2+) and other metal ions could not activate the lipase. According to the homology analysis and site-directed mutagenesis assay, the catalytic triad of the recombinant lipase was identified as Ser-165, Asp-260, and His-290 residues.

Topics: Amino Acid Sequence; Aspergillus fumigatus; Catalytic Domain; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Escherichia coli; Fungal Proteins; Gene Expression; Hydrogen-Ion Concentration; Industrial Microbiology; Kinetics; Lipase; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitrophenols; Plasmids; Recombinant Proteins; Sequence Analysis, DNA; Substrate Specificity; Temperature; Transformation, Bacterial

A series of poly(GMA-co-EGDMA) resins with identical composition but varying particle sizes, pore radii, specific surface areas and specific volumes are studied to assess how Candida antarctica lipase B immobilization affects the porosity of the copolymer particles. Mercury porosimetry reveals a significant change in the average pore size (up to 6.1-fold), the specific surface area (up to 3.2-fold) and the specific volume (up to 2.1-fold) of the epoxy resin. A similar behaviour is observed for glutaraldehyde-modified epoxy resins. The influences of the resin porosity properties on the loading of Candida antarctica lipase B during immobilization and on the hydrolytic activity (hydrolysis of p-nitrophenyl acetate) of the immobilized lipase are studied.

Topics: Adsorption; Biotechnology; Candida; Enzyme Stability; Enzymes, Immobilized; Epoxy Resins; Ethylene Glycols; Fungal Proteins; Glutaral; Hydrolysis; Lipase; Methacrylates; Nitrophenols; Particle Size; Porosity

Enzyme efficiency results from the cooperation of functional groups in the catalytic site. In order to mimic a natural enzyme, a definite 3D scaffold must be carefully designed so that the functional groups can work cooperatively. During the HIV-1 fusion process, the gp41 N- and C-terminal heptad repeat regions form a coiled-coil six-helical bundle (6HB) that brings the viral and target cell membranes into close proximity for fusion. We used 6HB as the molecular model for a novel scaffold for the design of an artificial enzyme, in which the modified C34 and N36 peptides formed a unique 6HB structure through specific molecular recognition, and the position and orientation of the side-chain groups on this scaffold were predictable. The histidine modified 6HB C34(H13/20)/N36(H15/22) showed enzyme-like hydrolytic activity towards p-nitrophenyl acetate (PNPA; k(cat)/K(M) =3.66 M(-1) s(-1)) through the cooperation of several inter- or intrahelical imidazole groups. Since the catalytic activity of 6HB depends on the C- and N-peptide assembly, either HIV fusion inhibitors that can compete with the formation of catalytic 6HB or denaturants that can destroy the ordered structure were able to modulate its activity. Further engineering of the solvent-exposing face with Glu(-)-Lys(+) salt bridges enhanced the helicity and the stability of 6HB. As a result, the population and stability of cooperative catalytic units increased. In addition, the Glu(-)-Lys(+) -stabilized 6HB SC35(H13/20)/N36(H15/22) had increased catalytic efficiency (k(cat)/K(M) =6.30 M(-1) s(-1)). A unique 6HB system was specifically assembled and provided a scaffold sufficiently stable to mimic the function of enzymes or other biomolecules.

Topics: Amino Acid Sequence; Biocatalysis; Biomimetic Materials; Circular Dichroism; HIV Envelope Protein gp41; HIV Fusion Inhibitors; HIV-1; Humans; Hydrolases; Hydrolysis; Kinetics; Molecular Sequence Data; Nitrophenols; Peptide Fragments; Protein Engineering; Protein Structure, Secondary

An Integrated Vacuum Carbonate Absorption Process (IVCAP) currently under development could significantly reduce the energy consumed when capturing CO2 from the flue gases of coal-fired power plants. The biocatalyst carbonic anhydrase (CA) has been found to effectively promote the absorption of CO2 into the potassium carbonate solution that would be used in the IVCAP. Two CA enzymes were immobilized onto three selected support materials having different pore structures. The thermal stability of the immobilized CA enzymes was significantly greater than their free counterparts. For example, the immobilized enzymes retained at least 60% of their initial activities after 90 days at 50 °C compared to about 30% for their free counterparts under the same conditions. The immobilized CA also had significantly improved resistance to concentrations of sulfate (0.4 M), nitrate (0.05 M) and chloride (0.3 M) typically found in flue gas scrubbing liquids than their free counterparts.

Topics: Absorption; Animals; Biotechnology; Carbon Dioxide; Carbonates; Carbonic Anhydrases; Cattle; Enzyme Stability; Enzymes, Immobilized; Hydrolysis; Nitrophenols; Porosity; Solutions; Temperature

Lactobacillus plantarum is an important lactic acid bacterium, usually found as natural inhabitant of food, such as fermented vegetables and meat products. However, little information about lactic acid bacteria, especially concerning L. plantarum, as a source of useful enzymes has been reported. The aim of this study was to clone, express in Escherichia coli, purify, and characterize an esterase from L. plantarum ATCC 8014. The esterase gene (1014 bp) was amplified and cloned in pET14b expression vector to express a His(6)-tagged protein in E. coli. Recombinant L. plantarum esterase was purified by Ni-NTA resin, presenting an apparent molecular mass of about 38 kDa. It presented highest activity at pH 6.0 and 40 degrees C. Also, it presented preference for p-nitrophenyl butyrate, but hydrolyzed more efficiently p-nitrophenyl acetate. Besides, this study shows, for the first time, CD data about secondary structure of an esterase from L. plantarum.

Topics: Amino Acid Sequence; Bacterial Proteins; Butyrates; Cloning, Molecular; DNA, Bacterial; Escherichia coli; Esterases; Genes, Bacterial; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Lactobacillus plantarum; Molecular Sequence Data; Molecular Weight; Nitrophenols; Plasmids; Protein Structure, Secondary; Recombinant Proteins; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Spectrophotometry, Ultraviolet; Substrate Specificity; Temperature

Carboxylesterase 1A1 (CES1A1) efficiently catalyses the hydrolysis of a substrate containing ester, amide, or thioester bonds. It is expressed at a high level in the human liver, but at a low level in the human kidney. In this study, we found the cause of this tissue-specific expression of the CES1A1 gene using 5-aza-2'-deoxycytidine (5-aza-dC) and bisulfite sequencing. Treatment of HEK293 cells, human embryonic kidney cells not expressing the CES1A1 gene, with 5-aza-dC caused dramatic expression of the CES1A1 gene. Bisulfite sequencing revealed that the region around the transcription start site (TSS) of the CES1A1 gene was almost entirely methylated in HEK293 cells, whereas the region was almost entirely unmethylated in HepG2 cells, human hepatoma cells. The hypomethylated DNA molecules for the region were observed in HEK293 cells treated with 5-aza-dC. In the genome obtained from the kidney, the region downstream of the TSS was methylated compared with that obtained from the liver. From these findings, it can be concluded that DNA methylation is involved in CES1A1 gene expression and that the difference between CES1A1 gene expression in the human kidney and that in the human liver may arise from the difference in DNA methylation levels in the region around the TSS.

Topics: Azacitidine; Base Sequence; Carboxylesterase; Cell Line; Decitabine; DNA Methylation; Gene Expression; Humans; Hydrolysis; Kidney; Liver; Molecular Sequence Data; Nitrophenols; RNA, Messenger; Transcriptional Activation

Adenylate cyclase-hemolysin toxin (CyaA) produced from the human respiratory tract pathogen Bordetella pertussis requires fatty-acyl modification by CyaC-acyltransferase to become an active toxin. Previously, the recombinant CyaA pore-forming (CyaA-PF) fragment expressed in Escherichia coli was shown to be hemolytically active upon palmitoylation in vivo by cosynthesized CyaC. Here, the 21-kDa CyaC enzyme separately expressed in E. coli as an inclusion body was solubilized in 8 M urea and successfully refolded into an enzymatically active monomer. In addition to the capability of activating CyaA-PF in vitro, CyaC showed esterase activity against p-nitrophenyl acetate (pNPA) and p-nitrophenyl palmitate (pNPP), with preferential hydrolysis toward pNPP when compared with chymotrypsin. A homology-based CyaC structure suggested a conceivable role of a catalytic triad including Ser(30), His(33) and Tyr(66) in substrate catalysis. Alanine substitutions of these individual residues caused a drastic decrease in specific activities of all three mutant enzymes (S30A, H33A and Y66A) toward pNPP, signifying that CyaC-acyltransferase shares a similar mechanism of hydrolysis with a serine esterase in which Ser(30) is part of the catalytic triad.

Topics: Acetyltransferases; Adenylate Cyclase Toxin; Amino Acid Sequence; Amino Acid Substitution; Bordetella pertussis; Catalytic Domain; Escherichia coli; Esterases; Gene Expression; Humans; Models, Chemical; Models, Molecular; Molecular Sequence Data; Molecular Weight; Mutagenesis, Site-Directed; Nitrophenols; Palmitates; Recombinant Proteins; Sequence Alignment; Substrate Specificity

Glutathione transferases (GSTs) are known as promiscuous enzymes capable of catalyzing the conjugation of glutathione with a broad range of electrophilic substrates. A previous study based on recombinant chimeras derived from human GST M1-1 and GST M2-2 demonstrated the formation of a subset of F1 generation GSTs, which had lost high activity with substrates distinguishing parental enzymes. In the present study, the members of this subset were recombined by DNA shuffling to produce an F2 generation of GSTs. Screening of 930 bacterial clones demonstrated that 83% of recombinant enzyme variants were active with at least one of three alternative substrates: phenethyl isothiocyanate (PEITC), 1-chloro-2,4-dinitrobenzene, or p-nitrophenyl acetate. The majority had similar low activity as the parental GSTs in the F1 generation. However, 17 novel enzymes displayed high activity with PEITC. Half of these enzymes were similar to GST M1-1, which also has high activity with the same substrate, and all of these GSTs featured Tyr116/Ser210 in the active site. This group of F2 variants apparently had reverted to the GST M1-1 type. A second group of F2 variants with high PEITC activity was characterized by His116 in the active site. This category represented a new variety of GSTs, which demonstrated higher selectivity for isothiocyanate substrates than the GST M1-1 type. The different groups of GSTs can be considered as distinct molecular quasi-species, each of which comprises variant amino acid sequences. The quasi-species are structurally distinguished by active-site residues that govern their substrate selectivities. Clearly, minimal alterations of the active site can generate enzymes with highly distinctive functional properties.

Topics: Bacterial Proteins; Catalytic Domain; Dinitrochlorobenzene; Glutathione Transferase; Humans; Isothiocyanates; Nitrophenols; Recombinant Proteins; Substrate Specificity

Drugs used clinically usually have a primary mechanism of action, but additional effects on other biological targets can contribute to their effects. A potentially useful additional target is the endocannabinoid metabolizing enzyme monoacylglycerol lipase (MGL). We have screened a range of drugs for inhibition of MGL and compared the observed potencies using different MGL enzyme assays.. MGL activity was screened using recombinant human MGL (cell lysates and purified enzyme) with 4-nitrophenyl acetate (NPA) as substrate. 2-Oleolyglycerol metabolism by rat cerebellar cytosolic MGL and by recombinant MGL was also investigated.. Among the 96 compounds screened in the NPA assay, troglitazone, CP55,940, N-arachidonoyl dopamine and AM404 inhibited NPA hydrolysis by the lysates with IC(50) values of 1.1, 4.9, 0.78 and 3.1µM, respectively. The potency for troglitazone is in the same range as its primary pharmacological activity, activation of peroxisome proliferator-activated receptor (PPAR) γ. Among PPARγ ligands, the potency order towards human MGL was troglitazone > ciglitazone > rosiglitazone > 15-deoxy-Δ(12,14) -prostaglandin J(2) ≈ CAY 10415 > CAY 10514. In contrast to the time-dependent inhibitor JZL184, the potency of troglitazone was dependent upon the enzyme assay system used. Thus, troglitazone inhibited rat cytosolic 2-oleoylglycerol hydrolysis less potently (IC(50) 41µM) than hydrolysis of NPA by the human MGL lysates.. 'Hits' in screening programmes for MGL inhibitors should be assessed in different MGL assays. Troglitazone may be a useful lead for the design of novel, dual action MGL inhibitors/PPARγ activators.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Modulators; Chromans; Cyclohexanols; Dopamine; Enzyme Assays; Glycerides; Humans; Monoacylglycerol Lipases; Nitrophenols; Piperidines; PPAR gamma; Rats; Rats, Sprague-Dawley; Rats, Wistar; Thiazolidinediones; Troglitazone

Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to α-, β-, γ- and ζ-classes and from various organisms, ranging from the bacteria, archaea to eukarya domains, were investigated for their esterase/phosphatase activity with 4-nitrophenyl acetate, 4-nitrophenyl phosphate and paraoxon as substrates. Only α-CAs showed esterase/phosphatase activity, whereas enzymes belonging to the β-, γ- and ζ-classes were completely devoid of such activity. Paraoxon, the metabolite of the organophosphorus insecticide parathione, was a much better substrate for several human/murine α-CA isoforms (CA I, II and XIII), with k(cat)/K(M) in the range of 2681.6-4474.9M(-1)s(-1), compared to 4-nitrophenyl phosphate (k(cat)/K(M) of 14.9-1374.4M(-1)s(-1)).

Topics: Animals; Carbon Dioxide; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Catalysis; Humans; Insecticides; Isoenzymes; Kinetics; Mice; Nitrophenols; Organophosphorus Compounds; Paraoxon; Substrate Specificity

Extracellular lipase from Bacillus coagulans BTS-3 was immobilized on (3 A x 1.5 mm) molecular sieve. The molecular sieve showed approximately 68.48% binding efficiency for lipase (specific activity 55 IU mg(-1)). The immobilized enzyme achieved approx 90% conversion of acetic acid and 4-nitrophenol (100 mM each) into 4-nitrophenyl acetate in n-heptane at 65 degrees C in 3 h. When alkane of C-chain length other than n-heptane was used as the organic solvent, the conversion of 4-nitrophenol and acetic acid was found to decrease. About 88.6% conversion of the reactants into ester was achieved when reactants were used at molar ratio of 1:1. The immobilized lipase brought about conversion of approximately 58% for esterification of 4-nitrophenol and acetic acid into 4-nitrophenyl acetate at a temperature of 65 degrees C after reuse for 5 cycles.

Topics: Acetic Acid; Bacillus; Biotechnology; Enzymes, Immobilized; Esters; Nitrophenols; Temperature

Carboxylesterases (CbEs) are key enzymes in pesticide detoxification. These esterases are involved in the biochemical mechanism for pesticide resistance in some pest species, and further they are considered an efficient protective mechanism against acute toxicity by organophosphate (OP) pesticides in mammals. To gain knowledge on the role of CbEs in pesticide toxicity and natural tolerance in earthworms, we performed an enzyme kinetic analysis to investigate whether these annelids are able to secrete them into their gut lumen. We determined levels of CbE activity and isozyme abundance in the gut wall and ingested soil collected from different portions of the gastrointestinal tract of Lumbricus terrestris. Moreover, modulation of enzyme activity by selected substrates (alpha-naphthyl acetate [alpha-NA], 4-nitrophenyl valerate [4-NPV] and 4-nitrophenyl acetate [4-NPA]) and OP pesticides was examined to compare the response between tissue and soil CbEs. We found a high CbE activity in the ingested soil extracts from the crop/gizzard (alpha-NA-CbE=8.43+/-2.76U mg(-1) protein and 4-NPA-CbE=5.98+/-2.11U mg(-1) protein) compared to the gut wall. Three lines of evidences suggest that the gut epithelium is the main source of this luminal CbE activity. First, the effect of substrate concentrations on CbE activity from both the ingested soil extracts and gut tissues resulted in similar apparent K(m) and V(max) values. Second, native PAGE gels revealed that some of the CbE isozymes in the gut tissue were also present in the soil extracts. Third, tissue and soil CbEs showed the same sensitivity to inhibition by OPs. The concentrations of insecticide causing 50% of esterase inhibition (IC(50)) was comparable between tissue (IC(50)s range=4.01-9.67nM dichlorvos and 8480-6880nM paraoxon) and soil (IC(50)s range=6.01-11.5nM dichlorvos and 8400-7260nM paraoxon). Our results suggest a set of (eco)toxicological implications and environmental applications derived from the ability of earthworms to secrete these pesticide-detoxifying enzymes.

Topics: Animals; Carboxylic Ester Hydrolases; Dichlorvos; Environmental Monitoring; Gastrointestinal Tract; Insecticides; Kinetics; Naphthols; Nitrobenzenes; Nitrophenols; Oligochaeta; Organophosphorus Compounds; Paraoxon; Soil Pollutants; Substrate Specificity; Valerates

Arylamine N-acetyltransferases (NATs) play an important role in both the detoxification of arylamine and hydrazine drugs and the activation of arylamine carcinogens. Because the catalytic triad, Cys-His-Asp, of mammalian NATs has been shown to be essential for maintaining protein stability, rendering it impossible to assess alterations of the triad on catalysis, we explored the impact of the highly conserved proximal residue, Tyr190, which forms a direct hydrogen bond interaction with one of the triad residues, Asp122, as well as a potential pi-pi stacking interaction with the active site His107. The replacement of hamster NAT2 Tyr190 by either Phe, Ile or Ala was well tolerated and did not result in significant alterations in the overall fold of the protein. Nevertheless, stopped-flow and steady-state kinetic analysis revealed that Tyr190 was critical for maximizing the acetylation rate of NAT2 and the transacetylation rate of p-aminobenzoic acid when compared with the wild-type. Tyr190 was also shown to play an important role in determining the pK(a) of the active site Cys during acetylation, as well as the pH versus the rate profile for transacetylation. We hypothesized that the pH dependence was associated with global changes in the active site structure, which was revealed by the superposition of [(1)H, (15)N] heteronuclear single quantum coherence spectra for the wild-type and Y190A. These results suggest that NAT2 catalytic efficiency is partially governed by the ability of Tyr190 to mediate the collective impact of multiple side chains on the electrostatic potential and local conformation of the active site.

Topics: Animals; Arylamine N-Acetyltransferase; Catalysis; Conserved Sequence; Cricetinae; Hydrogen-Ion Concentration; Kinetics; Mutagenesis, Site-Directed; Nitrophenols; Protein Conformation; Tyrosine

In this study, to explore the plasticity of the alpha/beta-hydrolase fold family, we converted bromoperoxidase A2 (BPO-A2) from Streptomyces aureofaciens to a lipase by structure comparison with lipase A (LipA) from Bacillus subtilis. These two enzymes have similar structures (2.1 A rmsd) and a very low level of sequence identity ( approximately 18%). A variant BL1 was constructed by deleting the caplike domain of BPO-A2 and further fine-tuning the newly formed substrate binding site. The lipase activity was successfully transplanted on BL1, while the halogenation activity was totally lost. BL1 also showed higher hydrolytic activities toward long chain p-nitrophenyl esters, such as p-nitrophenyl caprylate (3.7-fold) and p-nitrophenyl palmitate (7.0-fold), while its activity toward a short chain ester (p-nitrophenyl acetate) decreased dramatically, to only 1.2% of that of BPO-A2. After two rounds of directed evolution and site-directed mutagenesis on selected residues, several mutants with both improved hydrolytic activities and substrate preferences toward long chain substrates were obtained. The highest hydrolytic activity toward p-nitrophenyl palmitate of the best mutant BL1-2-E8-plusI was improved by 40-fold compared with that of BL1. These results demonstrate the possibility of manipulating the caplike domain of alpha/beta-hydrolase fold enzymes and provide further understanding of the structure-function relationship of the alpha/beta-hydrolase fold enzymes. The design strategy used in this study could serve as a useful approach for constructing variants with targeted catalytic properties using the alpha/beta-hydrolase fold.

Topics: Bacillus subtilis; Binding Sites; Biocatalysis; Caprylates; Esters; Hydrolysis; Lipase; Mutation; Nitrophenols; Palmitates; Peroxidases; Protein Conformation; Streptomyces aureofaciens; Substrate Specificity

Establishing esterase assays allows the determination and comparison of esteratic activities of tissues of one organism and between organisms. We have developed a high-performance liquid chromatography (HPLC) assay for the determination of S-acetylthiocholine (ATC) and p-nitrophenyl acetate (NPA) hydrolyzing activities of rat serum esterases based on ion pair chromatography with on-line radiochemical and ultraviolet (UV) detection. ATC is a substrate for cholinesterases, whereas NPA is cleaved by a variety of esterases and other proteins (e.g., cholinesterases, paraoxonase, carboxylesterase, albumin). Both substrates were incubated, simultaneously or separately, with rat serum to explore potential interferences between the enzymatic hydrolyses of the compounds. The ratio of the peak area of the (14)C-labeled substrates to the total peak area of the substrates and their corresponding cleavage products was compared with the UV quantitation of ATC and p-nitrophenolate (NP), the cleavage product of NPA, measured at 230 and 350 nm, respectively. The peak identity of ATC and NP was confirmed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The reaction rates of the assays using one substrate or both, as well as using radiochemical or UV detection, were equal. Moreover, the correlation between rat serum volumes and reaction rates was shown for both substrates. In conclusion, one can (i) choose between the two detection methods reliably, (ii) take advantage of monitoring both substrate and product by using radiochemical detection, and (iii) combine both substrates to determine esterase activities in rat serum and probably other biological matrices.

Topics: Acetylthiocholine; Animals; Chromatography, High Pressure Liquid; Chromatography, Liquid; Esterases; Female; Kinetics; Male; Mass Spectrometry; Nitrophenols; Rats; Rats, Wistar; Time Factors

The kinetics of alpha-chymotrypsin (alpha-CT) catalyzed hydrolysis of 4-nitrophenyl acetate has been studied in aqueous solution of alkyldimethylethanolammonium bromide (cetyl, dodecyl, decyl) surfactants at concentrations below and above their critical micelle concentration. From Michaelis-Mcnten kinetics, the catalytic rate constant kcat and the Michaelis constant KM have been determined. The bell-shaped profiles of alpha-CT activity with increasing surfactant concentrations indicate the interaction between the micelle-bound enzyme and substrate.

Topics: Biocatalysis; Chymotrypsin; Ethanolamine; Hydrolysis; Kinetics; Nitrophenols; Surface-Active Agents

The design of active sites has been carried out using quantum mechanical calculations to predict the rate-determining transition state of a desired reaction in presence of the optimal arrangement of catalytic functional groups (theozyme). Eleven versatile reaction targets were chosen, including hydrolysis, dehydration, isomerization, aldol, and Diels-Alder reactions. For each of the targets, the predicted mechanism and the rate-determining transition state (TS) of the uncatalyzed reaction in water is presented. For the rate-determining TS, a catalytic site was designed using naturalistic catalytic units followed by an estimation of the rate acceleration provided by a reoptimization of the catalytic site. Finally, the geometries of the sites were compared to the X-ray structures of related natural enzymes. Recent advances in computational algorithms and power, coupled with successes in computational protein design, have provided a powerful context for undertaking such an endeavor. We propose that theozymes are excellent candidates to serve as the active site models for design processes.

Topics: Acrolein; Aldehydes; Binding Sites; Catalysis; Cocaine; Enzyme Activation; Enzymes; Hydrolysis; Isomerism; Models, Molecular; Molecular Structure; Naphthols; Nitrophenols; Peptides; Proline; Quantum Theory; Sarin; Substrate Specificity; Water

The quasimonoclonal mouse is useful to examine B cell selection during T-dependent antibody (Ab) responses because of its limited B cell populations mainly expressing the knockin 17.2.25 V(H)-encoded H chain (V(H)T) paired with the lambda1 or lambda2 L chain. It has been reported that both two V(H)T/lambda1 and V(H)T/lambda2 B cell populations responded to a T-dependent antigen conjugated with a hapten p-nitrophenylacetyl (pNP), but only V(H)T/lambda2 B cells differentiated to secrete high affinity anti-pNP IgG Abs by acquiring a critical mutation (T313A) in the V(H)T. The V(H)T/lambda2 B cells may be more potent in migrating to the germinal centers (GCs) due to about 50-fold higher affinity for pNP than V(H)T/lambda1 B cells. Here, to uncover how V(H)T/lambda2 B cells were preferentially recruited for affinity maturation during the anti-pNP Ab response, we examined the L chain usage and mutation frequency of V(H)T(+) GC B cells at a single cell level. V(H)T/lambda2 B cells bearing the unmutated V(H)T gene were found in the GCs more frequently than V(H)T/lambda1 and mutated V(H)T/lambda2 counterparts in an early phase of the Ab response. In the course of the GC reaction, the number of V(H)T/lambda2 B cells that mutated their V(H)T genes preferentially expanded, and finally V(H)T/lambda2 B cells bearing the T313A mutation occupied V(H)T(+) GC B cell population. Thus, it is suggested that B cells with a higher affinity were selected not only for entry to the GCs but also in the affinity maturation process during a T-dependent Ab response.

Topics: Animals; Antibody Affinity; B-Lymphocytes; Base Sequence; Clone Cells; gamma-Globulins; Gene Rearrangement, B-Lymphocyte, Light Chain; Genes, Immunoglobulin Heavy Chain; Genes, Immunoglobulin Light Chain; Germinal Center; Haptens; Immunoglobulin lambda-Chains; Mice; Molecular Sequence Data; Mutation; Nitrophenols; T-Lymphocytes

The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.

Topics: Binding Sites; Calorimetry; Circular Dichroism; Esterases; Hydrogen-Ion Concentration; Kinetics; Ligands; Models, Molecular; Nitrophenols; Protein Binding; Protein Conformation; Pyruvaldehyde; Serum Albumin, Bovine; Thioctic Acid

Paraoxonase 1 (PON 1) is an enzyme that is promiscuous in its ability to hydrolyze various types of substrates. It hydrolyzes aryl esters, phosphate esters, lactones, and reduces lipid peroxides to hydroxides. Aspirin is an aryl ester with a short plasma half life. We hypothesized that aspirin would be effectively hydrolyzed by PON 1 and many of its anti-atherogenic effects, at least in part, could be accounted for by its antioxidant product, salicylic acid. In this study, we determined the ability of human plasma and PON 1-rich HDL to hydrolyze acetyl ester of salicylic acid (aspirin). The ability of aspirin to compete for the hydrolysis of paraoxon and p-nitrophenylacetate was determined. In addition, nitrated aspirin was synthesized and tested directly for hydrolysis. Aspirin competed for the hydrolysis of paraoxon and p-nitrophenylacetate by HDL in a dose-dependent manner. Human plasma and HDL were also able to hydrolyze nitroaspirin and aspirin and release nitrosalicylic acid and salicylic acid, respectively. These findings suggest that salicylic acid might be generated in the plasma from aspirin. The ability of long-term treatment with aspirin to retard atherosclerosis might be dependent on the generation of free salicylic acid, a scavenger of free radicals.

Topics: Adult; Antioxidants; Aryldialkylphosphatase; Aspirin; Atherosclerosis; Binding, Competitive; Biotransformation; Humans; Hydrolysis; In Vitro Techniques; Nitrophenols; Paraoxon; Platelet Aggregation Inhibitors; Salicylic Acid

A supramolecular inclusion complex, [Zn(L1)(H2O)2(beta-CD)](ClO4)2.9.5 H2O (1) was synthesized and characterized structurally and its first-order active species for hydrolysis of esters, [Zn(L1)(H2O)(OH)(beta-CD)](ClO4) (2), was isolated (L1=4-(4'-tert-butylbenzyl)diethylenetriamine; beta-CD=beta-cyclodextrin). The apparent inclusion stability constant of the host and the guest measured in aqueous solution was (5.91+/-0.03)x10(3) for 1. The measured values of the first- and second-order pK(a) values of coordinated water molecules were 8.20+/-0.08 and 10.44+/-0.08, respectively, and were assigned to water molecules occupying the plane and remaining axial positions in a distorted trigonal bipyramid of the [Zn(L1)(H2O)2(beta-CD)]2+ sphere according to the structural analysis of [Zn(L2)(H2O)}2(mu-OH)](ClO4)3 (3) (L2=4-benzyldiethylenetriamine). p-Nitrophenyl acetate (pNA) hydrolysis catalyzed by 1 at pH 7.5-9.1 and 25.0+/-0.1 degrees C exhibited a first-order reaction with various concentrations of pNA and 1, but the pH profile did not indicate saturated kinetic behavior. Second-order rate constants of 0.59 and 24.0 M(-1) s(-1) were calculated for [Zn(L1)(H2O)(OH)(beta-CD)]+ and [Zn(L1)(OH)2(beta-CD)], respectively; the latter exhibited a potent catalytic activity relative to the reported mononuclear and polynuclear Zn(II) species.

Topics: beta-Cyclodextrins; Binding Sites; Catalysis; Crystallography, X-Ray; Hydrolysis; Kinetics; Metalloendopeptidases; Models, Chemical; Molecular Mimicry; Molecular Structure; Nitrophenols; Oxidation-Reduction; Potentiometry; Water; Zinc Compounds

Previous mechanistic and crystallographic studies on two C-C hydrolase enzymes, Escherichia coli MhpC and Burkholderia xenovorans BphD, support a general base mechanism for C-C hydrolytic cleavage, rather than the nucleophilic mechanism expected for a serine hydrolase. The role of the active site serine residue could be to form a hydrogen bond with a gem-diolate intermediate, or to protonate such an intermediate. Hydrolase BphD is able to catalyse the hydrolysis of p-nitrophenyl benzoate ester substrates, which has enabled an investigation of these mechanisms using a Hammett analysis, and comparative studies upon five serine esterases and lipases from the alpha/beta-hydrolase family. A reaction parameter (rho) value of +0.98 was measured for BphD-catalysed ester hydrolysis, implying a build-up of negative charge in the transition state, consistent with a general base mechanism. Values of +0.31-0.61 were measured for other serine esterases and lipases, for the same series of esterase substrates. Pre-steady state kinetic studies of ester hydrolysis, using p-nitrophenyl acetate as the substrate, revealed a single step kinetic mechanism for BphD-catalysed ester hydrolysis, with no burst kinetics. A general base mechanism for BphD-catalysed ester hydrolysis is proposed, in which Ser-112 stabilises an oxyanion intermediate through hydrogen bonding, and assists the rotation of this oxyanion intermediate via proton transfer, a novel reaction mechanism for the serine catalytic triad.

Topics: Benzoates; Binding Sites; Burkholderia; Catalysis; Escherichia coli; Escherichia coli Proteins; Esterases; Esters; Hydrogen-Ion Concentration; Hydrolases; Hydrolysis; Kinetics; Lipase; Nitrophenols; Serine; Substrate Specificity

To determine the activity of a major intestinal esterase in the first-pass hydrolysis of O-isovaleryl-propranolol (isovaleryl-PL), a model ester compound, rat intestinal jejunum and blood vessels were perfused simultaneously after inhibition of a carboxylesterase (CES) by bis-p-nitrophenyl phosphate (BNPP). BNPP specifically inhibits approximately 90% of CES activity without influencing aminopeptidase activity or the transport of L-leucyl-p-nitroanilide and p-nitroaniline, nonester compounds. When isovaleryl-PL was perfused into the jejunal lumen after BNPP treatment, its absorption clearance (7.60 +/- 0.74 microl/min) increased approximately 3-fold compared with control, whereas its degradation clearance (32.5 +/- 5.40 microl/min) decreased to 23% of control. Therefore, CES seems to be mainly responsible for the intestinal first-pass hydrolysis of isovaleryl-PL. This finding is consistent with the results from studies of in vitro BNPP inhibition in the mucosal S9 fraction. V(max) values for valeryl-PL, isovaleryl-PL, and p-nitrophenyl acetate in the jejunal S9 fraction were 1.7- to 2.5-fold higher than that in the ileal S9 fraction, which agreed with the jejunum/ileum ratio (approximately 1.5-fold) of mRNA expression levels for the CES2 isozymes, AB010635 and AY034877. These findings indicated that CESs expressed in the intestine markedly contribute to first-pass hydrolysis in both jejunum and ileum.

Topics: Aminopeptidases; Anilides; Aniline Compounds; Animals; Biotransformation; Carboxylic Ester Hydrolases; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Hydrolysis; Ileum; Intestinal Absorption; Intestinal Mucosa; Isoenzymes; Jejunum; Male; Nitrophenols; Propranolol; Rats; Rats, Wistar; RNA, Messenger; Subcellular Fractions

This report describes a newly identified potential of grapefruit juice (GFJ) in mediating pharmacokinetic drug interactions due to its capability to inhibit esterase. The study demonstrates that GFJ inhibits purified porcine esterase activity toward p-nitrophenyl acetate and the prodrugs lovastatin and enalapril. In rat and human hepatic or gut S9 fractions and rat gut lumen, GFJ inhibited the hydrolysis of enalapril and lovastatin, which are known to be metabolized principally by esterases, lovastatin being metabolized also by CYP3A. In Caco-2 cells, with minimal CYP3A activity, permeability of these prodrugs was increased in the presence of GFJ. In rats, oral coadministration of GFJ or an esterase inhibitor, bis-(p-nitrophenylphosphate), with the prodrugs led to respective increases in plasma area under the curve by 70% or 57% for enalaprilat and 279% or 141% for lovastatin acid. In addition, portal vein-cannulated rats pretreated with GFJ at -15 and -2 h before lovastatin administration (10 mg/kg p.o.) as a solution, 1) in water and 2) in GFJ, showed, respectively, a 49% increase (CYP3A-inhibited) and a 116% increase (both CYP3A and gut esterase-inhibited) in the portal plasma exposure to the active acid, compared with a non-GFJ pretreatment group. Overall, along with the CYP3A inactivation by GFJ, the decreased esterase activity also played a significant role in increasing the metabolic stability and permeability of esters leading to enhancement of exposure to the active drugs in rats. These new esterase inhibition findings indicate that the potential of drug interaction between ester prodrugs and GFJ should also be considered in the clinic.

Topics: Administration, Oral; Angiotensin-Converting Enzyme Inhibitors; Animals; Beverages; Caco-2 Cells; Cell Membrane Permeability; Citrus paradisi; Enalapril; Enzyme Inhibitors; Esterases; Food-Drug Interactions; Fruit; Humans; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Injections, Intravenous; Intestinal Absorption; Intestines; Liver; Lovastatin; Male; Nitrophenols; Rats; Rats, Sprague-Dawley; Subcellular Fractions; Swine

Our previous studies described a newly identified potential of grapefruit juice (GFJ) in mediating pharmacokinetic drug interactions due to its capability of esterase inhibition. The current study identifies the active components in GFJ responsible for its esterase-inhibitory effect. The esterase-inhibitory potential of 10 constitutive flavonoids and furanocoumarins toward p-nitrophenylacetate (PNPA) hydrolysis was investigated. The furanocoumarins bergamottin, 6',7'-dihydroxybergamottin, and bergapten, and the glycoside flavonoids naringin and hesperidin, at concentrations found in GFJ or higher, did not inhibit the hydrolysis of PNPA by purified porcine esterase and human liver microsomes. However, the flavonoid aglycones morin, galangin, kaempferol, quercetin, and naringenin showed appreciable inhibition of PNPA hydrolysis in purified porcine esterase, and human and rat liver systems. In Caco-2 cells, demonstrated to contain minimal CYP3A activity, the permeability coefficient of the prodrugs lovastatin and enalapril was increased in the presence of the active flavonoids kaempferol and naringenin, consistent with inhibition of esterase activity. In rats, oral coadministration of kaempferol and naringenin with these prodrugs led to significant increases in plasma exposure to the active acids. In addition, in portal vein-cannulated rats, coadministration of lovastatin with kaempferol (10 mg/kg) led to a 154% and a 113% increase in the portal plasma exposure to the prodrug and active acid, respectively, compared with coadministration with water. The contribution of CYP3A inhibition was demonstrated to be minimal. Overall, a series of flavonoids present in GFJ are identified as esterase inhibitors, of which kaempferol and naringenin are shown to mediate pharmacokinetic drug interaction with the prodrugs lovastatin and enalapril due to their capability of esterase inhibition.

Topics: Administration, Oral; Angiotensin-Converting Enzyme Inhibitors; Animals; Beverages; Caco-2 Cells; Citrus paradisi; Dose-Response Relationship, Drug; Enalapril; Enzyme Inhibitors; Esterases; Flavanones; Flavonoids; Food-Drug Interactions; Fruit; Humans; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; In Vitro Techniques; Intestinal Absorption; Kaempferols; Lovastatin; Male; Microsomes, Liver; Nitrophenols; Rats; Rats, Sprague-Dawley

The rate of hydrolysis of p-nitrophenyl acetate (PNPA) catalyzed by Mucor javanicus lipase has been measured in AOT reverse micellar solutions formulated in aliphatic hydrocarbons, aromatic hydrocarbons and a chlorinated compound. The study has been performed at a single value of W=([water]/[AOT])=6.0. Fluorescence decay measurements of intrinsic enzyme fluorescence, mainly due to tryptophan residues, in the different reverse micellar systems were also carried out, in an attempt to obtain some insight on the effect of the organic solvent on the protein conformation. Differences observed in the kinetics of the fluorescence decays of tryptophan residues of the lipase incorporated to the micelles with the different external organic solvents were also found in the catalytic behaviour of the enzyme. In particular, it is observed that the contribution of the long lived component of the fluorescence decay is considerably higher (ca. 40%) in aliphatic than in aromatic solvents (ca. 15%), indicating significant differences in the protein conformation. This effect of the organic solvent on the protein conformation is also observed in the enzymatic activity, which is higher in the aromatic than in the aliphatic solvents.

Topics: Catalysis; Hydrolysis; Kinetics; Lipase; Micelles; Mucor; Nitrophenols; Organic Chemicals; Solvents; Succinates

Heme proteins--hemoglobin and myoglobin possess esterase activities. Studies with purified hemoglobin from normal individuals and diabetic patients revealed that the esterase activity as measured from hydrolysis of p-nitrophenyl acetate (p-NPA) was higher in diabetic condition and increased progressively with extent of the disease. HbA(1c), the major glycated hemoglobin, which increases proportionately with blood glucose level in diabetes mellitus, exhibited more esterase activity than the non-glycated hemoglobin fraction, HbA(0), as demonstrated spectrophotometrically as well as by activity staining. Glycation influenced esterase activity of hemoglobin by increasing the affinity for the substrate and the rate of the reaction. Both HbA(0) and HbA(1c)-mediated catalysis of p-NPA hydrolysis was pH-dependent. Esterase activity of in vitro-glycated myoglobin (GMb) was also higher than that of its non-glycated analog (Mb). The amplified esterase activities of hemoglobin and myoglobin might be associated with glycation-induced structural modifications of the proteins.

Topics: Adult; Animals; Blood Glucose; Diabetes Mellitus, Type 2; Esterases; Hemoglobins; Humans; Middle Aged; Myoglobin; Nitrophenols

Substrate specificity within the family of enzymes designated as homoserine transsuccinylases is variable, with some organisms utilizing succinyl-CoA and other organisms utilizing acetyl-CoA. In this study it is shown that the enzyme from Bacillus cereus uses acetyl-CoA as its acyl donor, but its catalytic rate is significantly lower than other HTS family members. BcHTS is inactivated by both iodoacetamide and diethyl pyrocarbonate and the enzyme can be partially protected from inactivation by the presence of succinyl-CoA. This leads to the conclusion that BcHTS can bind both acetyl-CoA and succinyl-CoA and suggests that it may represent an intermediate between the succinate-transferring HTS family members and the acetate-transferring HTS family members. The B. cereus enzyme was unable to rescue growth of an Escherichia coli strain lacking a functional transsuccinylase, however.

Topics: Bacillus cereus; Cloning, Molecular; Diethyl Pyrocarbonate; Enzyme Activation; Homoserine; Homoserine O-Succinyltransferase; Hydrogen-Ion Concentration; Iodoacetamide; Kinetics; Methionine; Mutant Proteins; Nitrophenols; Substrate Specificity

The capacity of human, minipig, and rat skin and liver subcellular fractions to hydrolyze the anesthetic ester procaine was compared with carboxylesterase substrates 4-methylumbelliferyl-acetate, phenylvalerate, and para-nitrophenylacetate and the arylesterase substrate phenylacetate. Rates of procaine hydrolysis by minipig and human skin microsomal and cytosolic fractions were similar, with rat displaying higher activity. Loperamide inhibited procaine hydrolysis by human skin, suggesting involvement of human carboxylesterase hCE2. The esterase activity and inhibition profiles in the skin were similar for minipig and human, whereas rat had a higher capacity to metabolize esters and a different inhibition profile. Minipig and human liver and skin esterase activity was inhibited principally by paraoxon and bis-nitrophenyl phosphate, classical carboxylesterase inhibitors. Rat skin and liver esterase activity was inhibited additionally by phenylmethylsulfonyl fluoride and the arylesterase inhibitor mercuric chloride, indicating a different esterase profile. These results have highlighted the potential of skin to hydrolyze procaine following topical application, which possibly limits its pharmacological effect. Skin from minipig used as an animal model for assessing transdermal drug preparations had similar capacity to hydrolyze esters to human skin.

Topics: Animals; Enzyme Inhibitors; Esterases; Esters; Female; Humans; Hydrolysis; Liver; Loperamide; Male; Microsomes, Liver; Molecular Structure; Nitrophenols; Paraoxon; Pentanoic Acids; Phenylacetates; Phenylmethylsulfonyl Fluoride; Procaine; Rats; Rats, Wistar; Skin; Swine; Swine, Miniature; Umbelliferones

The organophosphorus insecticides have been known for many years to cause cholinergic crisis in humans as a result of the inhibition of the critical enzyme acetylcholinesterase. The interactions of the activated, toxic insecticide metabolites (termed oxons) with acetylcholinesterase have been studied extensively for decades. However, more recent studies have suggested that the interactions of certain anticholinesterase organophosphates with acetylcholinesterase are more complex than previously thought since their inhibitory capacity has been noted to change as a function of inhibitor concentration. In the present report, chlorpyrifos oxon (O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphate) was incubated with human recombinant acetylcholinesterase in the presence of p-nitrophenyl acetate in order to better characterize kinetically the interactions of this oxon with enzyme. Determination of the dissociation constant, Kd, and the phophorylation rate constant, k2, for chlorpyrifos oxon with a range of oxon and p-nitrophenyl acetate concentrations revealed that Kd, but not k2, changed as a function of oxon concentration. Changes in p-nitrophenyl acetate concentrations did not alter these same kinetic parameters. The inhibitory capacity of chlorpyrifos oxon, as measured by ki (k2/Kd), was also affected as a result of the concentration-dependent alterations in binding affinity. These results suggest that the concentration-dependent interactions of chlorpyrifos oxon with acetylcholinesterase resulted from a different mechanism than the concentration-dependent interactions of acetylthiocholine. In the latter case, substrate bound to the peripheral anionic site of acetylcholinesterase has been shown to reduce enzyme activity by blocking the release of the product thiocholine from the active site gorge. With chlorpyrifos oxon, the rate of release of 3,5,6-trichloro-2-pyridinol is irrelevant since the active site is not available to interact with other oxon molecules after phosphorylation of Ser-203 has occurred.

Topics: Acetylcholinesterase; Acetylthiocholine; Binding Sites; Chlorpyrifos; Cholinesterase Inhibitors; Dose-Response Relationship, Drug; Humans; Hydrolysis; Insecticides; Kinetics; Models, Chemical; Nitrophenols; Phosphorylation; Recombinant Proteins; Serum Albumin, Bovine

Skin esterases serve an important pharmacological function as they can be utilised for activation of topically applied ester prodrugs. Understanding the nature of these enzymes, with respect to their role and local activity, is essential to defining the efficacy of ester prodrugs. Minipigs are used as models to study the kinetics of absorption of topically applied drugs. Their skin has structural properties very similar to human skin. However, regional distribution differences in esterase activity from site-to-site could influence cross-species extrapolation. Investigation of the regional site variation of minipig skin esterase activity will facilitate standardization of topically applied drug studies. Furthermore, the characterization of regional skin variation, will aid in translation of minipig results to better predictions of human esterase activity. Here we report the variation in rates of hydrolysis by minipig skin taken from different regional sites, using the esterase-selective substrates: phenyl valerate (carboxylesterase), phenyl acetate (arylesterase) and p-nitrophenyl acetate (general esterase). Skin from ears and back of male minipig showed higher activity than female. Skin from minipig ears and the back showed the highest level of esterase activity and was similar to human breast skin used in vitro absorption studies. These results suggest that skin from the minipig back is an appropriate model for preclinical human skin studies, particularly breast skin. This study supports the use of the minipig, with topical application to the back, as a model for the investigation of pharmacokinetics and metabolism of ester prodrugs.

Topics: Animals; Carboxylesterase; Carboxylic Ester Hydrolases; Cytosol; Drug Evaluation, Preclinical; Esterases; Female; Hydrolysis; Kinetics; Liver; Male; Microsomes; Models, Animal; Nitrophenols; Phenylacetates; Reproducibility of Results; Sex Factors; Skin; Substrate Specificity; Swine; Swine, Miniature; Valerates

With the overall objective of assessing the potential of utilizing plasma protein binding interactions in combination with the prodrug approach for improving the pharmacokinetics of drug substances, a series of model carbonate ester prodrugs of phenol, encompassing derivatives with fatty acid-like structures, were characterized in vitro. Stability of the derivatives was studied in aqueous solution, human serum albumin solution, human plasma, and rat liver homogenate at 37 degrees C. Stability of the derivatives in aqueous solution varied widely, with half-lives ranging from 31 to 1.7 x 10(4) min at pH 7.4 and 37 degrees C. The carbonate esters were subject to catalysis by plasma esterases except for the t-butyl and acetic acid derivatives, which were stabilized in both human plasma and human serum albumin solutions relative to buffer. In most cases, however, hydrolysis was accelerated in the presence of human serum albumin indicating that the derivatives interacted with the protein, a finding which was confirmed using the p-nitrophenyl acetate kinetic assay. Different human serum albumin binding properties of the phenol model prodrugs with fatty acid-like structure and neutral carbonate esters were observed. In the context of utilizing plasma protein binding in combination with the prodrug approach for optimizing drug pharmacokinetics, the esterase-like properties of human serum albumin towards the carbonate esters potentially allowing the protein to act as a catalyst of parent compound regenerations is interesting.

Topics: Animals; Carbonates; Drug Stability; Esterases; Esters; Fatty Acids; Humans; Hydrolysis; Molecular Structure; Nitrophenols; Phenol; Prodrugs; Protein Binding; Rats; Serum Albumin; Water

Human triacylglycerol hydrolase (hTGH) has been shown to play a role in hepatic lipid metabolism. Triacylglycerol hydrolase (TGH) hydrolyzes insoluble carboxylic esters at lipid/water interfaces, although the mechanism by which the enzyme adsorbs to lipid droplets is unclear. Three-dimensional modeling of hTGH predicts that catalytic residues are adjacent to an alpha-helix that may mediate TGH/lipid interaction. The helix contains a putative neutral lipid binding domain consisting of the octapeptide FLDLIADV (amino acid residues 417-424) with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino acid except proline) identified in several other proteins that bind or metabolize neutral lipids. Deletion of this alpha-helix abolished the lipolytic activity of hTGH. Replacement of F417 with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L418 and L420 with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C390 is available for covalent acylation. However, neither chemical modification of C390 nor mutation to alanine affected activity. Our findings indicate that F417 but not L418, L420, or C390 participates in substrate hydrolysis by hTGH.

Topics: Acylation; Amino Acid Sequence; Animals; Binding Sites; Butyrates; Catalysis; Cell Line; Chlorocebus aethiops; COS Cells; Cysteine; Gene Deletion; Gene Expression; Humans; Hymecromone; Iodoacetamide; Lipase; Mercaptoethanol; Mutagenesis, Site-Directed; Mutation; Nitrophenols; Phenylalanine; Point Mutation; Protein Folding; Recombinant Proteins; Sequence Homology, Amino Acid; Spodoptera; Substrate Specificity; Transfection

The kinetics of base-catalyzed hydrolysis of 4-nitrophenyl acetate was studied in water and water-ethanol mixtures under ultrasound (21.1 kHz) using a probe equipped with a quartz horn and, by comparison, without sonication. The ethanol concentration was varied from 0 to 50 wt % and pH from 7.5 to 9. The increase of reaction rate was inversely proportional to the strength of substrate interactions with the solvent structure. It was found that the effect of ultrasound could be related to the perturbation of solute-solvent interactions. This conclusion seems to apply to both acid-catalyzed and base-catalyzed hydrolysis reactions. Parallel experiments with a titanium horn revealed the catalytic action of undetermined chemical species formed from dispersed titanium metal.

Topics: Catalysis; Ethanol; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Molecular Structure; Nitrophenols; Titanium; Ultrasonics

Malaria control in southern Mozambique is currently by indoor residual carbamate insecticide treatment, with pyrethroid-treated bed-nets distributed to pregnant women and children under five in northern Mozambique. The susceptibility of Anopheles funestus s.s. to pyrethroid, carbamate, organochlorine, and organophosphorus insecticides was determined by World Health Organization adult mosquito susceptibility tests at 19 localities in Mozambique, from March 2000 to July 2002. Biochemical assays were carried out on mosquitoes from the same families to detect shifts in the quantity or activity of enzyme families involved in insecticide detoxification. An. funestus from all localities remained fully susceptible to DDT and the organophosphorus insecticide malathion. A high level of pyrethroid resistance was detected in An. funestus populations in southern Mozambique. An. funestus outside Maputo province were still susceptible to pyrethroids. An. funestus from six localities also were resistant to carbamate insecticides propoxur and bendiocarb. Both pyrethroid and carbamate resistance occurred in five of these six localities. Mosquitoes from five of the localities with elevated p450 estimates, compared with the insecticide-susceptible Durban strain, were pyrethroid-resistant. The only exception to this trend was Mozal, which had elevated p450 estimates but full pyrethroid susceptibility by bioassay. The lack of cross-resistance between pyrethroids and DDT in Mozambican An. funestus suggests that a kdr-type target site resistance mechanism has not been selected. Low levels of insecticide-insensitive acetylcholinesterase, the target site for carbamates and organophosphates, were found in all populations tested. The high level of metabolically based pyrethroid resistance has implications for current malaria control programs in Mozambique.

Topics: Acetylcholinesterase; Animals; Anopheles; Carbamates; Cytochrome P-450 Enzyme System; DDT; Esterases; Female; Insect Vectors; Insecticide Resistance; Insecticides; Malathion; Male; Mortality; Mozambique; Nitrophenols; Pyrethrins

In the degradation of chlorophyll, chlorophyllase catalyzes the initial hydrolysis of the phytol moiety from the pigment. Since chlorophyll degradation is a defining feature of plant senescence, compounds inhibiting chlorophyllase activity may delay senescence, thereby improving shelf life and appearance of plant products. Here we describe the development of a 96-well plate-based purification and assay system for measuring chlorophyllase activity. Integrated lysis and immobilized metal affinity chromatography plates were used for purifying recombinant hexahistidine-tagged Triticum aestivum (wheat) chlorophyllase from Escherichia coli. Chlorophyllase assays using chlorophyll as a substrate showed that the immobilized fusion protein displayed kinetic parameters similar to those of recombinant enzyme purified by affinity chromatography; however, the need to extract reaction products from a multiwell plate limits the value of this assay for high-throughput screening applications. Replacing chlorophyll with p-nitrophenyl-ester substrates eliminates the extraction step and allows for continuous measurement of chlorophyllase activity in a multiwell plate format. Determination of steady state kinetic constants, pH rate profile, the inhibitory effects of metal ions and esterase inhibitors, and the effect of functional group-modifying reagents validated the utility of the plate-based system. The combined purification and assay system provides a convenient and rapid method for the assessment of chlorophyllase activity.

Topics: Biological Assay; Carboxylic Ester Hydrolases; Chlorophyll; Chromatography, Affinity; Escherichia coli; Genes, Plant; Histidine; Hydrogen-Ion Concentration; Kinetics; Nitrophenols; Oligopeptides; Recombinant Fusion Proteins; Triticum

Screening of a "one-bead-one-compound" peptide library containing biomimetic His/Cys ligands has led to the discovery of sequences that hydrolyze ester substrates in combination with Zn2+.

Topics: Amino Acid Sequence; Binding Sites; Combinatorial Chemistry Techniques; Cysteine; Histidine; Hydrolysis; Ligands; Metalloproteins; Molecular Sequence Data; Molecular Structure; Nitrophenols; Peptide Library; Peptides; Sequence Analysis, Protein; Time Factors; Zinc

Many topically applied drugs contain esters that are hydrolyzed in the skin. Minipigs have emerged as potential models of human dermatology and, in some aspects, may be superior to commonly used rat skin. The aims of this study were to evaluate the suitability of minipig and rat skin as in vitro models of human epidermal esterase activity.. Naphthyl acetate and para-nitrophenyl acetate were tested as prototypical substrates of carboxylesterases from skin, plasma, and liver. Reaction products were monitored by high-performance liquid chromatography/ultraviolet analysis.. Hydrolysis efficiency in skin was higher than plasma, but lower than liver. The esterase efficiency of rat skin microsomes (580-1100 min(-1) mg(-1)) was two to three orders of magnitude higher than human (1.3-4.2 min(-1) mg(-1)) and minipig microsomes (1.2-4.2 min(-1) mg(-1)). Rat skin cytosol (80-100 min(-1) mg(-1)) was 2- to 10-fold more efficient than human (2.4-67 min(-1) mg(-1)) or minipig cytosol (18-61 min(-1) mg(-1)). Most importantly, human skin fractions displayed kinetics of hydrolysis very similar to minipig skin.. These studies show minipig skin as an appropriate, potentially valuable model for human epidermal ester metabolism and support the use of minipig skin in preclinical development of topically applied compounds.

Topics: Acetylcholinesterase; Animals; Blood; Carboxylesterase; Cholinesterase Inhibitors; Humans; In Vitro Techniques; Kinetics; Liver; Male; Naphthols; Neostigmine; Nitrophenols; Phenotype; Rats; Rats, Sprague-Dawley; Skin; Species Specificity; Swine; Swine, Miniature

Human carbonic anhydrase II (CA II), a zinc metalloenzyme, was screened against 960 structurally diverse, biologically active small molecules. The assay monitored CA II esterase activity against the substrate 4-nitrophenyl acetate in a format allowing high-throughput screening. The assay proved to be robust and reproducible with a hit rate of approximately 2%. Potential hits were further characterized by determining their IC(50) and K(d) values and tested for nonspecific, promiscuous inhibition. Three known sulfonamide CA inhibitors were identified: acetazolamide, methazolamide, and celecoxib. Other hits were also found, including diuretics and antibiotics not previously identified as CA inhibitors, for example, furosemide and halazone. These results confirm that many sulfonamide drugs have CA inhibitory properties but also that not all sulfonamides are CA inhibitors. Thus many, but not all, sulfonamide drugs appear to interact with CA II and may target other CA isozymes. The screen also yielded several novel classes of nonsulfonamide inhibitors, including merbromin, thioxolone, and tannic acid. Although these compounds may function by some nonspecific mechanism (merbromin and tannic acid), at least 1 (thioxolone) appears to represent a genuine CA inhibitor. Thus, this study yielded a number of potentially new classes of CA inhibitors and preliminary experiments to characterize their mechanism of action.

Topics: Carbonic Anhydrase II; Carbonic Anhydrase Inhibitors; Drug Evaluation, Preclinical; Enzyme Stability; Esterases; Humans; Inhibitory Concentration 50; Kinetics; Nitrophenols; Octoxynol; Reproducibility of Results; Structure-Activity Relationship; Substrate Specificity; Sulfonamides

Random mutagenesis followed by a filter-based screening assay has been used to identify a mutant of human class 1 aldehyde dehydrogenase (ALDH1) that was no longer inhibited by Mg(2+) ions but was activated in their presence. Several mutants possessed double, triple, and quadruple amino acid substitutions with a total of seven different residues being altered, but each had a common T244S change. This point mutation proved to be responsible for the Mg(2+) ion activation. An ALDH1 T244S mutant was recombinantly expressed and was used for mechanistic studies. Mg(2+) ions have been shown to increase the rate of deacylation. Consistent with the rate-limiting step for ALDH1 being changed from coenzyme dissociation to deacylation was finding that chloroacetaldehyde was oxidized more rapidly than acetaldehyde. Furthermore, Mg(2+) ions only in the presence of NAD(H) increased the rate of hydrolysis of p-nitrophenyl acetate showing that the metal only affects the binary complex. Though the rate-limiting step for the T244S mutant was different from that of the native enzyme, the catalytic efficiency of the mutant was just 20% that of the native enzyme. The basis for the change in the rate-limiting step appears to be related to NAD(+) binding. Using the structure of a sheep class 1 ALDH, it was possible to deduce that the interaction between the side chain of T244 and its neighboring residues with the nicotinamide ring of NAD(+) were an essential determinant in the catalytic action of ALDH1.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Animals; Catalysis; Cations, Divalent; Computational Biology; Cytosol; Enzyme Activation; Esterases; Humans; Hydrolysis; Isoenzymes; Magnesium; Models, Molecular; Mutagenesis; NAD; Nitrophenols; Point Mutation; Protein Conformation; Retinal Dehydrogenase; Sheep

The western flower thrips, Frankliniella occidentalis (Pergande), is a serious pest in the south-east of Spain owing to its direct feeding on crops, transmission of the tomato spotted wilt virus and its very high level of resistance to insecticides. Mechanisms of resistance were examined using field populations of F. occidentalis with different susceptibilities to acrinathrin, methiocarb (selective insecticides), endosulfan, metamidophos and deltamethrin (broad-spectrum insecticides). Esterase activity towards alpha-naphthyl acetate and p-nitrophenyl acetate in resistant strains was significantly higher than in the reference strain (MLFOM) for both model substrates. This higher activity was significantly correlated with acrinathrin and methiocarb resistance.

Topics: Animals; Esterases; Insecta; Insecticide Resistance; Insecticides; Lethal Dose 50; Methiocarb; Naphthols; Nitrophenols; Pyrethrins

Two strains of bacteria were isolated from deep-ocean sediments of the South China Sea using enrichment culturing technique and they were identified as Sphingomonas yanoikuyae DOS01 (AY878409) and Variovorax paradoxus T4 (AY878410) based on 16S rRNA gene sequences. S. yanoikuyae DOS01 was only capable of transforming dimethyl terephthalate (DMTP) to monomethyl terephthalate (MMTP) without further degradation while V. paradoxus T4 exhibited ability in mineralizing DMTP as the sole source of carbon and energy. The biochemical pathway of DMTP degradation was through MMTP and terephthalic acid (TA) as major detectable degradation intermediates in the culture media by both microorganisms. V. paradoxus T4 utilized DMTP and MMTP via hydrolysis of diester and monoester in the initial steps in degradation as confirmed by total organic carbon analysis of the culture medium and esterase activity assay of the lysed cells and fraction. The specific hydrolysis activity of esterase induced by DMTP or MMTP showed that greater hydrolysis of p-nitrophenyl acetate by esterase induced by DMTP-grown cells than that induced by MMTP. Results of this research suggest that the cleavage of the two identical carboxylic ester groups of phthalate diester are carried out by highly specific esterases of the same bacteria in the environment.

Topics: Carbon; Chemistry, Organic; Geologic Sediments; Hydrolysis; Kinetics; Models, Chemical; Molecular Sequence Data; Nitrophenols; Oceans and Seas; Phthalic Acids; Phylogeny; Proteobacteria; RNA, Ribosomal, 16S; Soil Microbiology; Sphingomonas

The effect of modulating the shape and the size of the hydrophobic pocket on the esterase activity and specificity of human carbonic anhydrase II (HCAII) for esters with different acyl chain lengths was investigated. Following an initial screen of 7 HCAII variants with alanine substitutions in positions 121, 143 and 198, detailed kinetic measurements were performed on HCAII and the variants V121A, V143A and V121A/V143A. For some variants, an increased size of the hydrophobic pocket resulted in increased activities and specificities for longer substrates. For V121A/V143A, the rate of hydrolysis for paranitrophenyl valerate was increased by a factor of approximately 3000. The specificities also changed dramatically, for example V121A/V143A is 6.3 times more efficient with paranitrophenyl valerate than paranitrophenyl acetate, while HCAII is >500 times more efficient with paranitrophenyl acetate than paranitrophenyl valerate. An automated docking procedure was performed on these variants with transition state analogues (TSAs) for the hydrolysis reaction. It was possible to correlate the catalytic rate constants to the docking results, i.e. for each variant, efficient hydrolysis was generally correlated to successful TSA-docking. The observations in this paper show that the redesign increased the catalytic rates for substrates with long acyl chains by removal of steric hinders and addition of new favourable binding interactions.

Topics: Alanine; Amino Acid Substitution; Binding Sites; Carbonic Anhydrase II; Catalysis; Esterases; Esters; Humans; Hydrolysis; Hydrophobic and Hydrophilic Interactions; Nitrobenzenes; Nitrophenols; Protein Conformation; Substrate Specificity; Valerates

A 120 member library of peptidocalix[4]arenes was synthesized and screened for catalysis of the hydrolysis of p-nitrophenyl acetate. His-Ser-His-calix[4]arene was found to catalyze this reaction with v(0)=3.24 x 10(-8)M/s, an increase of 1520% above background and 30% above the tripeptide (His-Ser-His) alone.

Topics: Bridged-Ring Compounds; Calixarenes; Catalysis; Hydrolysis; Models, Chemical; Nitrophenols; Oligopeptides; Phenols; Time Factors

Water-in-oil microemulsions are thermodynamically stable single-phase dispersions of water and surfactant within a continuous oil phase. The classical ternary system, based on the surfactant sodium bis(2-ethylhexyl)sulfosuccinate ('AOT'), water and an alkane such as n-heptane, is an optically transparent monodispersion of spherical water-droplets coated with a close-packed surfactant monolayer and the droplet radius is, to a good first approximation, directly proportional to the molar water: surfactant ratio, R. Enzymes dissolved in the water droplets retain activity and stability. These systems have attracted interest as media for biotransformations. Principally based upon studies in AOT-stabilized w/o microemulsions, a peculiar feature of the kinetics of enzyme-catalyzed reactions has long been apparent: the reaction rate characteristically increases from around zero at R=3, through a maximum, in the range R= 10-20, and thereafter decreases again, so that plots of rate vs. R are characteristically 'bell-shaped'. Furthermore, at optimal R, enzymes seem to be 'hyperactive', i.e., they are more active, by a modest but significant factor of 2-3-fold, than in aqueous solution. In this paper we propose the hypothesis that this kind of R-dependence arises because of the presence of freely mobile lone surfactant counterions (Na+) within the water-pool. These ions have no charge partners within the water pool and consequently have a high electrochemical potential. According to our model, lone counterions facilitate the hydrolysis of ester or amide substrates, for example, by stabilizing the tetrahedral intermediate formed during the reaction through ion-pairing with the carbonyl oxygen of the substrate, thus facilitating transfer of negative charge from the carbonyl carbon as it is attacked by the incoming nucleophile. An expression for the relationship between the concentration of free counterions in the water-pool and the compositional parameter R leads directly, through Debye-Hückel theory, to an expression for the relationship between the reaction rate and R, log k(R)= log k(o) + C(1/R)1/2 where k(R) is the rate constant at some finite R, k(o) is the rate constant extrapolated to R = infinity and C is an R-independent coefficient. For enzymes that display bell-shaped kinetics, such as bovine alpha-chymotrypsin and Chromobacterium viscosum lipase, the descending part of the plot (i.e. from optimal R to high R) obeys this equation very well. Inspection of the

Topics: Arylsulfonates; Buffers; Chymotrypsin; Dioctyl Sulfosuccinic Acid; Emulsions; Enzyme Activation; Glycylglycine; Kinetics; Lipase; Nitrophenols; Osmolar Concentration; Surface-Active Agents; Taurine; Water

An extracellular lipase was purified from the fermentation broth of Bacillus coagulans ZJU318 by CM-Sepharose chromatography, followed by Sephacryl S-200 chromatography. The lipase was purified 14.7-fold with 18% recovery and a specific activity of 141.1 U/mg. The molecular weight of the homogeneous enzyme was (32 kDa), determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme activity was maximum at pH 9.0 and was stable over a pH range of 7.0-10.0, and the optimum temperature for the enzyme reaction was 45 degrees C. Little activity loss (6.2%) was observed after 1 h of incubation at 40 degrees C. However, the stability of the lipase decreased sharply at 50 and 60 degrees C. The enzyme activity was strongly inhibited by Ag+ and Cu2+, whereas EDTA caused no inhibition. SDS, Brij 30, and Tween-80 inhibited lipase, whereas Triton X-100 did not significantly inhibit lipase activity.

Topics: Bacillus; Chromatography, Gel; Chromatography, Ion Exchange; Detergents; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Hydrogen-Ion Concentration; Lipase; Nitrophenols; Palmitates; Salts; Temperature

Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-L-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K(m) and V(max) values for the same substrate by Lineweaver-Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K(m) values at optimum pH and 20 degrees C were 0.625 mM, 0.541 mM, 0.785 mM and 0.862 mM with p-nitro phenyl acetate, for all CA localizations. The respective V(max) values at optimum pH and 20 degrees C were 0.875 micromol/L min, 0.186 micromol/L min, 0.214 micromol/L min and 0.253 micromol/L min with the same substrate. The K(i) and I50 values for the inhibitors sulphanilamide, KSCN, NaN3 and acetazolamide were determined for all the CA localizations.

Topics: Acetazolamide; Amides; Animals; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Cattle; Chromatography, Affinity; Cytosol; Electrophoresis, Polyacrylamide Gel; Kinetics; Nitrophenols; Sodium Azide; Stomach; Thiocyanates; Zinc

Twelve single nucleotide polymorphisms (SNPs) in the human CES2 gene, which encodes a carboxylesterase, hCE-2 [human carboxylesterase 2 (EC 3.1.1.1)], have been reported in the Japanese. In this report, we have examined functional alterations of three SNPs, a nonsynonymous SNP (100C>T, R34W), an SNP at the splice acceptor site in intron 8 (IVS8-2A>G), and one newly discovered nonsynonymous SNP (424G>A, V142M). For the two nonsynonymous SNPs, the corresponding variant cDNAs were expressed in COS-1 cells. Both the R34W and V142M variants showed little esterase activities toward the anticancer agent irinotecan and two typical carboxylesterase substrates, p-nitrophenol acetate and 4-methylumbelliferyl acetate, although increased levels of cDNA-mediated protein expression were observed by Western blotting as compared with the wild type. To investigate a possible splicing aberration in IVS8-2A>G, an in vitro splicing assay was utilized and transcripts derived from CES2 gene fragments of the wild type and IVS8-2A>G were compared. Sequence analysis of the cloned transcripts revealed that IVS8-2A>G yielded mostly aberrantly spliced transcripts, including a deleted exon or a 32-bp deletion proximal to the 5' end of exon 9, which resulted in truncated hCE-2 proteins. These results suggested that 100C>T (R34W), 424G>A (V142M), and IVS8-2A>G are functionally deficient SNPs.

Topics: Animals; Asian People; Camptothecin; Carboxylesterase; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Humans; Irinotecan; Nitrophenols; Polymorphism, Single Nucleotide; RNA Splicing; Umbelliferones

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from AcCoA to primary arylamines, hydrazines, and hydrazides and play a very important role in the metabolism and bioactivation of drugs, carcinogens, and other xenobiotics. The reaction follows a ping-pong bi-bi mechanism. Structure analysis of bacterial NATs revealed a Cys-His-Asp catalytic triad that is strictly conserved in all known NATs. Previously, we have demonstrated by kinetic and isotope effect studies that acetylation of the hamster NAT2 is dependent on a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)) and not a general acid-base catalysis. In addition, we established that, after formation of the acetylated enzyme intermediate, the active-site imidazole, His-107, is likely deprotonated at physiological pH. In this paper, we report steady-state kinetic studies of NAT2 with two acetyl donors, acetyl coenzyme A (AcCoA) and p-nitrophenyl acetate (PNPA), and four arylamine substrates. The pH dependence of k(cat)/K(AcCoA) exhibited two inflection points at 5.32 +/- 0.13 and 8.48 +/- 0.24, respectively. The pK(a) at 5.32 is virtually identical with the previously reported pK(a) of 5.2 for enzyme acetylation, reaffirming that the first half of the reaction is catalyzed by a thiolate-imidazolium ion pair in the active site. The inflection point at 8.48 indicates that a pH-sensitive group on NAT2 is involved in AcCoA binding. A Brønsted plot constructed by the correlation of log k(4) and log k(H)2(O) with the pK(a) for each arylamine substrate and water displays a linear free-energy relationship in the pK(a) range from -1.7 (H(2)O) to 4.67 (PABA), with a slope of beta(nuc) = 0.80 +/- 0.1. However, a further increase of the pK(a) from 4.67 (PABA) to 5.32 (anisidine) resulted in a 2.5-fold decrease in the k(4) value. Analysis of the pH-k(cat)/K(PABA) profile revealed a pK(a) of 5.52 +/- 0.14 and a solvent kinetic isotope effect (SKIE) of 2.01 +/- 0.04 on k(cat)/K(PABA). Normal solvent isotope effects of 4.8 +/- 0.1, 3.1 +/- 0.1, and 3.2 +/- 0.1 on the k(cat)/K(b) for anisidine, pABglu, and PNA, respectively, were also determined. These observations are consistent with a deacetylation mechanism dominated by nucleophilic attack of the thiol ester for arylamines with pK(a) values or=5.5. The general base is likely His-107 because the His-107 to Gln and Asn mutants were found to be devoid of catal

Topics: Acetyl Coenzyme A; Amines; Amino Acid Substitution; Animals; Arylamine N-Acetyltransferase; Carcinogens; Catalysis; Cricetinae; Hydrazines; Hydrogen-Ion Concentration; Kinetics; Nitrophenols; Protein Binding; Xenobiotics

Reactivators of acetylcholinesterase (AChE; EC 3.1.1.7) are able to treat intoxication by organophosphorus compounds, especially with pesticides or nerve agents. Owing to the fact that there exists no universal "broad-spectrum" reactivator of organophosphates-inhibited AChE, many laboratories have synthesized new AChE reactivators. Here, we synthesized five new and three previously known quaternary monopyridinium oximes as potential reactivators of AChE inhibited by nerve agents. Potencies to cleave p-nitrophenyl acetate (PNPA), which is commonly used as a model substrate of nerve agents, were measured. Their cleaving potencies were compared with 4-PAM (4-hydroxyiminomethyl-1-methylpyridinium iodide), which is derived from the structure of the currently used AChE-reactivator 2-PAM (2-hydroxyiminomethyl-1-methylpyridinium iodide). Three newly synthesized oximes achieved similar nucleophilicity at the similar pKa according to 4-PAM, which is very promising for using these derivatives as AChE reactivators.

Topics: Acetylcholinesterase; Cholinesterase Reactivators; Drug Synergism; Kinetics; Ligands; Molecular Structure; Nitrophenols; Oximes; Pralidoxime Compounds; Pyridinium Compounds

The specific activity of lipase A (Aspergillus niger) toward the hydrolysis of p-nitrophenyl acetate (p-NPA) is shown to increase as a result of sodium salt addition according to specific ion effects of the Hofmeister series. This shows explicitly that the Hofmeister effect is due to the different specific interactions between anions and the enzymatic surface.

Topics: Anions; Aspergillus niger; Buffers; Fungal Proteins; Hydrogen-Ion Concentration; Hydrolysis; Lipase; Nitrophenols

Topics: Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Catalysis; Combinatorial Chemistry Techniques; Dipeptides; Drug Evaluation, Preclinical; Hydrolysis; Molecular Structure; Nitrophenols; Peptide Library; Protein Binding; Sulfonamides

Mass spectrometry (MS)-based techniques have enormous potential for kinetic studies on enzyme-catalyzed processes. In particular, the use of electrospray ionization (ESI) MS for steady-state measurements is well established. However, there are very few reports of MS-based studies in the pre-steady-state regime, because it is difficult to achieve the time resolution required for this type of experiment. We have recently developed a capillary mixer with adjustable reaction chamber volume for kinetic studies by ESI-MS with millisecond time resolution (Wilson, D. J.; Konermann, L. Anal. Chem. 2003, 75, 6408-6414). Data can be acquired in kinetic mode, where the concentrations of selected reactive species are monitored as a function of time, or in spectral mode, where entire mass spectra are obtained for selected reaction times. Here, we describe the application of this technique to study the kinetics of enzyme reactions. The hydrolysis of p-nitrophenyl acetate by chymotrypsin was chosen as a simple chromophoric model system. On-line addition of a "makeup solvent" immediately prior to ionization allowed the pre-steady-state accumulation of acetylated chymotrypsin to be monitored. The rate constant for acetylation, as well as the dissociation constant of the enzyme-substrate complex obtained from these data, is in excellent agreement with results obtained by conventional stopped-flow methods. Bradykinin was chosen to illustrate the performance of the ESI-MS-based method with a nonchromophoric substrate. In this case, the unfavorable rate constant ratio for acylation and deacylation of the enzyme precluded measurements in the pre-steady-state regime. Steady-state experiments were carried out to determine the turnover number and the Michaelis constant for bradykinin. The methodologies used in this work open a wide range of possibilities for future ESI-MS-based kinetic assays in enzymology.

Topics: Acetylation; Bradykinin; Chymotrypsin; Hydrolysis; Kinetics; Nitrophenols; Spectrometry, Mass, Electrospray Ionization; Time Factors

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to arylamines, hydrazines, and their N-hydroxylated arylamine metabolites. The recently determined three-dimensional structures of prokaryotic NATs have revealed a cysteine protease-like Cys-His-Asp catalytic triad, which resides in a deep and hydrophobic pocket. This catalytic triad is strictly conserved across all known NATs, including hamster NAT2 (Cys-68, His-107, and Asp-122). Treatment of NAT2 with either iodoacetamide (IAM) or bromoacetamide (BAM) at neutral pH rapidly inactivated the enzyme with second-order rate constants of 802.7 +/- 4.0 and 426.9 +/- 21.0 M(-1) s(-1), respectively. MALDI-TOF and ESI mass spectral analysis established that Cys-68 is the only site of alkylation by IAM. Unlike the case for cysteine proteases, no significant inactivation was observed with either iodoacetic acid (IAA) or bromoacetic acid (BAA). Pre-steady state and steady state kinetic analysis with p-nitrophenyl acetate (PNPA) and NAT2 revealed a single-exponential curve for the acetylation step with a second-order rate constant of (1.4 +/- 0.05) x 10(5) M(-1) s(-1), followed by a slow linear rate of (7.85 +/- 0.65) x 10(-3) s(-1) for the deacetylation step. Studies of the pH dependence of the rate of inactivation with IAM and the rate of acetylation with PNPA revealed similar pK(a)(1) values of 5.23 +/- 0.09 and 5.16 +/- 0.04, respectively, and pK(a)(2) values of 6.95 +/- 0.27 and 6.79 +/- 0.25, respectively. Both rates reached their maximum values at pH 6.4 and decreased by only 30% at pH 9.0. Kinetic studies in the presence of D(2)O revealed a large inverse solvent isotope effect on both inactivation and acetylation of NAT2 [k(H)(inact)/k(D)(inact) = 0.65 +/- 0.02 and (k(2)/K(m)(acetyl))(H)/(k(2)/K(m)(acetyl))(D) = 0.60 +/- 0.03], which were found to be identical to the fractionation factors (Phi) derived from proton inventory studies of the rate of acetylation at pL 6.4 and 8.0. Substitution of the catalytic triad Asp-122 with either alanine or asparagine resulted in the complete loss of protein structural integrity and catalytic activity. From these results, it can be concluded that the catalytic mechanism of NAT2 depends on the formation of a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)). However, in contrast to the case with cysteine proteases, a pH-dependent protein conformational change is likely responsible for the second pK(a), and not deprotonati

Topics: Acetamides; Acetyl Coenzyme A; Acetylation; Alkylating Agents; Amino Acid Sequence; Amino Acid Substitution; Animals; Arylamine N-Acetyltransferase; Binding Sites; Cricetinae; Cysteine; Deuterium; Enzyme Inhibitors; Hydrogen-Ion Concentration; Iodoacetamide; Isoenzymes; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitrophenols; Papain; Protein Conformation; Recombinant Proteins

The aim of this study was to elucidate the absorption mechanism in Caco-2 and rat intestine models in order to improve the accuracy of prediction of oral absorption of ester prodrugs. Pivampicillin and cefcapene pivoxil hydrochloride (CFPN-PI), ester-type oral antibiotics, were chosen as model ester prodrugs. The level of esterase activity in Caco-2 cells was lower than that measured in the rat jejunum when p-nitrophenyl acetate was used as a substrate. Almost complete ester hydrolysis occurred before the ester prodrugs reached the basolateral side of the monolayer, and the disappearance of prodrugs was thought to be due to metabolism or transport after addition to the apical side of the monolayer. When pivampicillin and CFPN-PI were used, the amounts of ampicillin and cefcapene (CFPN) produced by hydrolysis of prodrugs were increased because intracellular degradation of prodrugs resulted in intracellular accumulation. On the other hand, when ampicillin or CFPN was used, only a small amount of the drug reached the basolateral side of the monolayers and no intracellular accumulation was observed. The permeability of CFPN-PI, the solubility of which is dependent on the acidity of gastric juice, across a Caco-2 monolayer or rat intestine, was also investigated by using an in vitro system that mimics the physiological state of the human gastrointestinal tract. The oral absorption of CFPN-PI in humans is predicted to be good either in the Caco-2 model or in the rat intestine model. It is concluded that our system may be a valuable tool for evaluation of oral absorption of ester prodrugs metabolized during permeation through the intestinal epithelium. Broader evaluation of such a system is warranted.

Topics: Animals; Caco-2 Cells; Cephalosporins; Epithelial Cells; Escherichia coli; Esters; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Hydrogen-Ion Concentration; Intestinal Absorption; Intestinal Mucosa; Intestines; Male; Nitrophenols; Prodrugs; Rats; Rats, Wistar; Serratia; Spectrophotometry, Ultraviolet; Staphylococcus aureus

Neisseria meningitidis serogroup A capsular polysaccharide (CPS) is composed of a homopolymer of O-acetylated, alpha1-->6-linked ManNAc 1-phosphate that is distinct from the capsule structures of the other meningococcal disease-causing serogroups, B, C, Y, and W-135. The serogroup A capsule biosynthetic genetic cassette consists of four open reading frames, mynA-D (sacA-D), that are specific to serogroup A, but the functions of these genes have not been well characterized. mynC was found to encode an inner membrane-associated acetyltransferase that is responsible for the O-acetylation of the CPS of serogroup A. The wild-type CPS as revealed by 1H NMR had 60-70% O-acetylated ManNAc residues that contained acetyl groups at O-3, with some species acetylated at O-4 and at both O-3 and O-4. A non-polar mynC mutant generated by introducing an aphA-3 kanamycin resistance cassette produced CPS with no O-acetylation. A serogroup A capsule-specific monoclonal antibody was shown to recognize the wild-type O-acetylated CPS, but not the CPS of the mynC mutant, which lacked O-acetylation. MynC was C-terminally His-tagged and overexpressed in Escherichia coli to obtain the predicted approximately 26-kDa protein. The acetyltransferase activity of purified MynC was demonstrated in vitro using [14C]acetyl-CoA. MynC O-acetylated the O-acetylated CPS of the mynC mutant and further acetylated the wild-type CPS of serogroup A meningococci, but not the CPS of serogroup B or C meningococci. Genetic complementation of the mynC mutant confirmed the function of MynC as the serogroup A CPS O-3 and O-4 acetyltransferase. MynC represents a new subclass of O-acetyltransferases that utilize acetyl-CoA to decorate the D-mannosamine capsule of N. meningitidis serogroup A.

Topics: Acetyltransferases; Antibodies, Monoclonal; Bacterial Capsules; Cell Membrane; Chromatography; Cytosol; Databases as Topic; Dose-Response Relationship, Drug; Drug Resistance; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Genetic Complementation Test; Hexosamines; Hydrogen-Ion Concentration; Immunoblotting; Kanamycin; Magnetic Resonance Spectroscopy; Models, Genetic; Mutation; Neisseria meningitidis; Nitrophenols; Polymerase Chain Reaction; Polymers; Polysaccharides, Bacterial; Promoter Regions, Genetic; Protein Structure, Tertiary; Subcellular Fractions; Time Factors

Earlier, we have shown that rat hepatic and pancreatic fatty acid ethyl ester (FAEE) synthases are structurally and functionally similar to rat liver carboxylesterase (CE) and pancreatic cholesterol esterase (ChE), respectively. We have also reported that only hepatic FAEE synthase is inhibited by tri-o-tolylphosphate (TOTP) in vivo and in human hepatocellular carcinoma (HepG2) cells. The metabolism of TOTP is a prerequisite for the inhibition of hepatic FAEE synthase as well as esterase activity. To further elucidate the mechanism of such differential inhibition by inhibitors of serine esterases, we synthesized two metabolites of TOTP, 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one (CBDP; cyclic saligenin phosphate) and di-o-tolyl-o-( proportional, variant -hydroxy)tolylphosphate (HO-TOTP), and one ChE inhibitor, 3-benzyl-6-chloro-2-pyrone (3-BCP). The inhibitory effect of CBDP, HO-TOTP, and 3-BCP on FAEE synthase and esterase activity was studied using rat hepatic and pancreatic postnuclear (PN) fractions, commercial porcine hepatic CE and pancreatic ChE, and in HepG2 and rat pancreatic tumor (AR42J) cell lines. Only HO-TOTP and CBDP inhibited FAEE synthase as well as esterase activity of hepatic PN fraction and commercial CE and ChE in a concentration-dependent manner, and the inhibition was found to be irreversible. However, no inhibition was found in pancreatic PN fraction by both TOTP metabolites and 3-BCP. Although 3-BCP inhibited only the esterase activity of commercial ChE in a concentration-dependent manner, the activity was reversible within 30 min of incubation. Studies with HepG2 cells also showed a significant inhibition of FAEE synthase-esterase activity by CBDP and HO-TOTP within 15 min of incubation, while no inhibition was observed in AR42J cells. 3-BCP did not inhibit FAEE synthase-esterase activity either in HepG2 or AR42J cells. Such differential inhibitory effect of the TOTP metabolites on hepatic and pancreatic FAEE synthase-esterase is supported by our earlier in vivo and in vitro studies. Further investigations are needed to understand the biochemical mechanism(s) of inactivation of TOTP metabolites and 3-BCP in the pancreas and AR42J cells towards FAEE synthase-esterase activities.

Topics: Acyltransferases; Animals; Carboxylesterase; Cell Line, Tumor; Cholinesterases; Esterases; Humans; Indicators and Reagents; Liver; Microdialysis; Nitrophenols; Organophosphorus Compounds; Pancreas; Pyrones; Serine; Substrate Specificity; Swine

To investigate the properties of unnatural zinc finger peptides with CysHis(3)-type ligand combinations, the HCHH- and CHHH-type zinc finger proteins (zf(HCHH) and zf(CHHH), respectively) were created by mutating Cys to His in the Cys(2)His(2)-type zinc finger of the transcription factor Sp1 (zf(CCHH)). The CD measurements clearly show that the single-finger CysHis(3)-type zinc finger peptides (zf(HCHH)f2 and zf(CHHH)f2) are folded by complexation with Zn(II). From the gel mobility shift assays, the CysHis(3)-type zinc finger proteins (zf(HCHH) and zf(CHHH)) evidently bind to the GC-box DNA, though the DNA binding affinity is lower than that of the wild CCHH-type zinc finger protein. Furthermore, the zf(HCHH)f2 and zf(CHHH)f2 peptides catalyze the hydrolysis of the 4-nitrophenyl acetate in contrast to the catalytically inactive zf(CCHH) peptide. This is the first study of the CysHis(3)-type zinc finger proteins and also provides useful information for redesigning artificial metalloproteins.

Topics: Amino Acid Sequence; Base Composition; Base Sequence; Binding Sites; Circular Dichroism; Cysteine; Electrophoretic Mobility Shift Assay; Histidine; Hydrolysis; Kinetics; Molecular Sequence Data; Nitrophenols; Peptide Fragments; Protein Structure, Tertiary; Sp1 Transcription Factor; Spectrophotometry, Ultraviolet; Zinc Fingers

The sensitivity of an enzyme to its environment has provoked much interest both for its immediate relevance to biochemistry and for the use of enzymes in chemical synthesis. The intercellular or extracellular environment in which an enzyme naturally operates is crowded with macromolecular, small-molecule, and ionic solutes and hence is markedly different from the dilute aqueous buffer solutions commonly cited for comparisons of biochemical processes. We report the results of a kinetic study into the effects of such a crowded solution on the rate of an enzyme-mediated process-the trypsin-catalyzed hydrolysis of a nonnatural substrate ester. The catalytic rate constant decreases linearly with solvent polarity, but substrate binding is independent of the concentration of added crowding agent up to 395 g/L.

Topics: Acetoacetates; Amides; Biomimetic Materials; Cytosol; Hydrolysis; Kinetics; Nitrophenols; Polyethylene Glycols; Trypsin

A novel zinc(II) hydroxide complex with a rare alkylthiolate donor in the coordination sphere is formed in aqueous solution from the dissolution of the zinc alkyl precursor complex (PATH)ZnCH(3) (PATH = 2-methyl-1-[methyl(2-pyridin-2-ylethyl)amino]propane-2-thiolate) in H(2)O and protonolysis of the Zn-C bond to give (PATH)ZnOH (1). The (PATH)ZnOH complex has been shown to promote the hydrolysis of 4-nitrophenyl acetate (4-NA) by a detailed kinetic study and is the first functional model for the zinc form of the enzyme peptide deformylase. From a fit of the sigmoidal pH-rate profile a kinetic pK(a) of 8.05(5) and a pH-independent second-order rate constant (k" max)) of 0.089(3) M(-1) s(-1) have been obtained. The kinetic pK(a) is similar to the pK(a) of 7.7(1) determined by a potentiometric study (25 degrees C, I = 0.1 (NaNO3)). Observation of both rate enhancement and turnover shows that 1 acts as a catalyst for the hydrolysis of 4-NA, although the turnovers are modest. Activation parameters have been obtained from a temperature-dependence study of the rate constants (E(a) = 54.8 kJ mol(-1), DeltaH++ = 52.4 kJ mol(-1), and DeltaS++ = -90.0 J mol(-1) K(-1)), and support a reaction mechanism which depends on nucleophilic attack of 1 in the rate-determining step. This is the first kinetic and thermodynamic study of a 4-coordinate zinc hydroxide complex containing a thiolate donor. In addition it is only the second time that a complete set of activation parameters have been obtained for the zinc-promoted hydrolysis of a carboxylic ester. This study puts the basicity and nucleophilicity of a (N(2)S)ZnOH complex in context with those of other L(n)()ZnOH complexes and enzymes.

Topics: Amidohydrolases; Catalysis; Hydrogen-Ion Concentration; Hydrolysis; Indicators and Reagents; Magnetic Resonance Spectroscopy; Nitrophenols; Organometallic Compounds; Potentiometry; Spectrophotometry, Ultraviolet; Sulfhydryl Compounds; Temperature; Zinc

Formation of fatty acid ethyl esters (FAEEs, catalyzed by FAEE synthase) has been implicated in the pathogenesis of chronic pancreatitis. In previous studies, we demonstrated that FAEE synthase, purified from rat liver microsomes, is identical to rat liver carboxylesterase (pI 6.1), and structurally and functionally different than that from pancreas. In this study, we purified and characterized rat pancreatic microsomal FAEE synthase, and determined its relationship with rat pancreatic cholesterol esterase (ChE). Since most of the serine esterases express p-nitrophenyl acetate (PNPA)-hydrolyzing activity as well as synthetic activity to form fatty acid esters or amides with a wide spectrum of alcohols and amines, respectively, we used PNPA-hydrolyzing activity to monitor the purification of FAEE synthase during various chromatographic purification steps. Synthesizing activity towards FAEEs, fatty acid methyl esters, and fatty acid anilides was measured only in the pooled fractions. At each step of purification (ammonium sulfate saturation, Q Sepharose XL, and heparin-agarose column chromatographies, and high performance liquid chromatography (anion exchange and gel filtration)) synthetic as well as hydrolytic activities copurified. Using ethanol, methanol, or aniline as substrates, the ester or anilide synthesizing activity of the purified protein was found to be 8709, 13000, and 2201 nmol/h/mg protein, respectively. The purified protein displayed a single band with an estimated molecular mass of approximately 68 kD upon SDS-PAGE under reduced denaturing conditions, cross-reacted with antisera against rat pancreatic ChE and showed 100% N-terminal sequence homology of the first 15 amino acids to that of rat pancreatic ChE. These results suggest that the purified protein has broad substrate specificity towards the conjugation of endogenous long chain fatty acids with substrates having hydroxyl and amino groups and is identical to ChE.

Topics: Acyltransferases; Ammonium Sulfate; Animals; Blotting, Western; Chemical Precipitation; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Fatty Acids; Hydrolysis; Microsomes; Molecular Weight; Nitrophenols; Pancreas; Rats; Sequence Analysis, Protein; Sterol Esterase; Swine

A directed evolution with phage-displayed random polypeptides of about 140 amino acid residues was followed until the sixth generation under a selection based on affinity to a transition state analog for an esterase reaction. The experimental design deliberately limits the observation to only 10 clones per generation. The first generation consists of three soluble random polypeptides and seven arbitrarily chosen clones from a previously constructed library. The clone showing the highest affinity in a generation was selected and subjected to random mutagenesis to generate variants for the next generation. Even within only 10 arbitrarily chosen polypeptides in each of the generations, there are enough variants in accord to capacity of binding affinity. In addition, the binding capacity of the selected polypeptides showed a gradual continuous increase over the generation. Furthermore, the purified selected random polypeptides exhibited a gradual but significant increase in esterase activity. The ease of the functional development within a small sequence variety implies that enzyme evolution is prompted even within a small population of random polypeptides.

Topics: Amino Acid Sequence; Bacteriophage M13; Cloning, Molecular; Directed Molecular Evolution; Esterases; Gene Library; Genetic Variation; Hydrolysis; Kinetics; Molecular Sequence Data; Mutagenesis; Nitrophenols; Peptide Library; Peptides; Protein Binding; Substrate Specificity

An ester hydrolase (ABL) has been isolated from a strain of Arthrobacter species (RRLJ-1/95) maintained in the culture collection of this laboratory. The purified enzyme has a specific activity of 1700 U/mg protein and is found to be composed of a single subunit (Mr 32,000), exhibiting both lipase and esterase activities shown by hydrolysis of triglycerides and p-nitrophenyl acetate respectively. Potential application of the enzyme concerns the asymmetrisation of prochiral 2-benzyl-1,3-propanediol esters besides enantioselective hydrolysis of alkyl esters of unsubstituted and substituted 1-phenyl ethanols.

Topics: Arthrobacter; Bacterial Proteins; Carboxylic Ester Hydrolases; Hydrolysis; Lipase; Molecular Weight; Nitrophenols; Propanols; Stereoisomerism; Triglycerides

Among substances which may prove useful in preventing or reducing the progression of glycooxidative modifications of proteins, heparin plays a unique role. To elucidate the mechanism whereby heparin may favourably influence the protein structure during glycation, human serum albumin (HSA) was glycated with both 25 and 50 mM glucose in the absence and presence of 12 microg.mL(-1) low-molecular-mass heparin. Glycation caused: (a) modifications of fluorescence emission and excitation spectra consistent with the covalent attachment of glucose to protein; (b) a significant increase in the esterase activity of HSA on p-nitrophenyl acetate; (c) a reduced susceptibility to tryptic digestion and (d) enhanced formation of high-molecular mass aggregates of HSA. These alterations were accompanied by oxidative reactions, as the EPR spectra showed a clear-cut radical signal, dependent on glucose concentration, further confirmed by measurement of the carbonyl content of HSA, as an indirect proof of oxidative damage. In the presence of heparin all the above alterations, especially at 25 mM glucose, turned out to be antagonized. The effects of heparin were dependent on its specific binding to HSA, which triggered an oxidative mechanism strikingly different from that caused by glucose. In the presence of heparin, only the radical species catalyzed by heparin was detected across all samples of glycated HSA, irrespective of glucose concentration. In addition, at 25 mM glucose, enhancement of the oxidative capacity of heparin was also observed. The results demonstrate that the oxidative mechanism sustained by heparin mediates biological effects that may be beneficial in reducing the extent of glycooxidative damage on HSA.

Topics: Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Esterases; Free Radicals; Glucose; Heparin; Humans; Hyperglycemia; Nitrophenols; Oxygen; Serum Albumin; Spectrophotometry; Time Factors; Trypsin

The presented pH-dyn assay serves as a versatile tool for screening enzymatically catalyzed reactions consuming or producing acids. The method is based on material balances of substrates and products. Ion balances relate concentrations of acids and bases to pH. pH-changes caused by the enzymatically catalyzed reaction in a well-defined buffer system are recorded by light-absorption measurements of a pH-indicator. Kinetic parameters are estimated by fitting the modeled pH changes to the experimentally observed ones. The enzymatically catalyzed hydrolysis of 4-nitrophenol is used as a model system. A pH indicator, bromothymol blue, is used to monitor the reaction progress. The reaction is monitored until the limiting substrate is completely consumed. This allows the estimation of the parameters of the Michaelis-Menten kinetics, K(M) and k(cat), in a single run. The results agree well with conventional spectrophotometric experiments and values reported in literature. Around pH 7, environmental CO(2) influences pH. Carbon dioxide influence was included in the model. Thus it was possible to estimate initial CO(2) concentrations as a model parameter, and therefore automatic correction for the CO(2) disturbances was achieved. This was important to detect low conversions at low buffer concentrations.

Topics: Calibration; Carbon Dioxide; Chemistry Techniques, Analytical; Hydrogen-Ion Concentration; Hydrolases; Hydrolysis; Kinetics; Models, Chemical; Nitrophenols

An esterase hydrolyzing phthalate esters has been purified from Micrococcus sp. YGJ1. The enzyme, a monomeric protein (Mr = 56 kDa) with a pI of 4.0, hydrolyzes various aliphatic and aromatic carboxylesters. The medium chain (C3-C4) esters are the most preferred substrates. The enzyme is inhibited by HgCl2 and p-chloromercuribenzoate but not by phenylmethylsulfonyl fluoride.

Topics: Dibutyl Phthalate; Enzyme Inhibitors; Esterases; Hydrolysis; Isoelectric Point; Kinetics; Mercuric Chloride; Micrococcus; Molecular Weight; Nitrophenols; p-Chloromercuribenzoic Acid; Phthalic Acids; Substrate Specificity

Mimics of carboxypeptidase A, a prototypical metalloprotease, were synthesized by linking macrocyclicpolyamines to the primary side of beta-cyclodextrin followed by complexing with Zn(II). These enzyme mimics exhibit saturation kinetics in hydrolysis of p-nitrophenyl acetate (PNPA) and enhance the rate of hydrolysis reaction by almost 300-fold. The effective molarities (EM) of the mimics range from 0.2 to 1.9 M. Origin of the rate acceleration was examined: the reactivity of Zn(II) complexes of [12]aneN3 [12]aneN4, and [14]aneN4 for hydrolyzing PNPA increases with increase in basicity of the zinc bound hydroxides [Zn(II)-OH], yielding a linear Brönsted plot. Free hydroxide fits well on this plot. A similar plot was obtained with the enzyme mimics. The Brönsted relationships indicate that the Zn(II)-OH in the catalytic systems hydrolyzes the ester by direct nucleophilic attack on the ester carbonyl of cyclodextrin-bound but not Zn(II)-coordinated PNPA.

Topics: beta-Cyclodextrins; Binding Sites; Carboxypeptidases; Catalysis; Cyclodextrins; Esters; Hydrolysis; Kinetics; Metalloendopeptidases; Models, Chemical; Models, Molecular; Molecular Mimicry; Molecular Structure; Nitrophenols; Polyamines; Zinc

Directed evolution of p-nitrobenzyl esterase (pNB E) has yielded eight generations of increasingly thermostable variants. The most stable esterase, 8G8, has 13 amino acid substitutions, a melting temperature 17 degrees C higher than the wild-type enzyme, and increased hydrolytic activity toward p-nitrophenyl acetate (pNPA), the substrate used for evolution, at all temperatures. Room-temperature activities of the evolved thermostable variants range from 3.5 times greater to 4.0 times less than wild type. The relationships between enzyme stability, catalytic activity, and flexibility for the esterases were investigated using tryptophan phosphorescence. We observed no correlation between catalytic activity and enzyme flexibility in the vicinity of the tryptophan (Trp) residues. Increases in stability, however, are often accompanied by decreases in flexibility, as measured by Trp phosphorescence. Phosphorescence data also suggest that the N- and C-terminal regions of pNB E unfold independently. The N-terminal region appears more thermolabile, yet most of the thermostabilizing mutations are located in the C-terminal region. Mutational studies show that the effects of the N-terminal mutations depend on one or more mutations in the C-terminal region. Thus, the pNB E mutants are stabilized by long-range, cooperative interactions between distant parts of the enzyme.

Topics: Directed Molecular Evolution; Dithionitrobenzoic Acid; Enzyme Stability; Esterases; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Luminescent Measurements; Models, Molecular; Mutation; Nitrophenols; Pliability; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Temperature; Tryptophan; Vibration

In order to select a species for drug absorption studies of ester prodrugs and to identify a possible absorption window with low esterase activity and hence increased absorption of the ester prodrug, the esterase activity was investigated in homogenates from various intestinal segments of different species. p-Nitrophenyl acetate and tenofovir disoproxil [bis(POC)-PMPA] were used as substrates for esterases. p-Nitrophenyl acetate is a model substrate for esterase activity, while tenofovir disoproxil (fumarate salt) is an ester prodrug of the potent antiviral nucleoside phosphonate analogue tenofovir. As esterase-mediated degradation during transepithelial transport may be a limiting factor for its oral absorption, targeting the prodrug to a region of the intestine with lower esterase activity may lead to an increase in oral absorption of the prodrug. The results obtained with p-nitrophenyl acetate and tenofovir disoproxil showed both a site-specific (duodenum > or = jejunum > ileum > or = colon) and species-dependent (rat > man > pig) degradation in intestinal homogenates. Degradation of tenofovir disoproxil in homogenates from Caco-2 monolayers (0.016+/-0.003 nmol. s(-1). mg protein(-1)) was low compared to its degradation in homogenates from human ileum (0.177+/-0.052 nmol. s(-1). mg protein(-1)). Rat ileum appears to be a suitable model to evaluate the influence of esterase activity on the oral absorption of the ester prodrug, as the degradation rate for tenofovir disoproxil (0.245+/-0.054 nmol. s(-1). mg protein(-1)) in rat ileum was similar to degradation in human ileum. The results also suggest that colon targeting may be a useful strategy to reduce the esterase-mediated degradation of ester prodrugs, hence resulting in a possible increase in their oral absorption.

Topics: Animals; Caco-2 Cells; Esters; Humans; Intestinal Mucosa; Male; Nitrophenols; Prodrugs; Rats; Species Specificity; Swine

Irinotecan (CPT-11) is a topoisomerase I inhibitor commonly used in the treatment of colorectal tumors. It is a prodrug, converted to an active metabolite, SN-38, by carboxylesterases (CEs). CEs are ubiquitary enzymes that react with numerous substrates. A specific CPT-11 converting enzyme was isolated from rat serum, with different kinetic properties than other CEs. We determined kinetic properties of specific CPT-11 CE activity (CPT-CE) in human normal liver and colon tumors. Km were very similar (3.4 microM in liver and 3.8 microM in colon tumors), but Vmax was higher in liver (2.7 pmol/min/mg protein) than in colon tumor (1.7 pmol/min/mg protein). CPT-CE and total CE (using p-nitro-phenylacetate as substrate) were weakly correlated in colon tumors. The large interpatient variability observed in liver CPT-CE activity could play a potential role in the pharmacokinetic variability observed with irinotecan.

Topics: Animals; Antineoplastic Agents, Phytogenic; Camptothecin; Carboxylic Ester Hydrolases; Colon; Colonic Neoplasms; DNA Topoisomerases, Type I; Enzyme Activation; Enzyme Inhibitors; Humans; Irinotecan; Kinetics; Liver; Nitrophenols; Rats; Substrate Specificity; Topoisomerase I Inhibitors

The effect of the low-molecular-mass natural reagents in high concentrations is important for investigating enzymatic reactions in near "in vivo" conditions and for optimisation of biotechnology processes. A model system, including p-nitrophenyl acetate as substrate and alpha-chymotrypsin as proteolytic enzyme, has been used to study the effect of high concentrations of sucrose, both influencing the viscosity of the reaction medium and acting as a nucleophilic effector (activator) on the enzymatic reaction. A kinetic scheme at high concentrations of nucleophilic effectors (sucrose) has been proposed.

Topics: Animals; Cattle; Chymotrypsin; Hydrolysis; In Vitro Techniques; Indicators and Reagents; Kinetics; Nitrophenols; Substrate Specificity; Sucrose

Penicillin G acylase catalysed the hydrolysis of 4-nitrophenyl acetate with a kcat of 0.8 s-1 and a Km of 10 microM at pH 7.5 and 20 degreesC. Results from stopped-flow experiments fitted a dissociation constant of 0.16 mM for the Michaelis complex, formation of an acetyl enzyme with a rate constant of 32 s-1 and a subsequent deacylation step with a rate constant of 0.81 s-1. Non-linear Van't Hoff and Arrhenius plots for these parameters, measured at pH 7.5, may be partly explained by a conformational transition affecting catalytic groups, but a linear Arrhenius plot for the ratio of the rate constant for acylation relative to KS was consistent with energy-compensation between the binding of the substrate and catalysis of the formation of the transition state. At 20 degreesC, the pH-dependence of kcat was similar to that of kcat/Km, indicating that formation of the acyl-enzyme did not affect the pKa values (6.5 and 9.0) of an acidic and basic group in the active enzyme. The heats of ionization deduced from values of pKa for kcat, which measures the rate of deacylation, are consistent with alpha-amino and guanidinium groups whose pKa values are decreased in a non-polar environment. It is proposed that, for catalytic activity, the alpha-amino group of the catalytic SerB1 and the guanidinium group of ArgB263 are required in neutral and protonated states respectively.

Topics: Acylation; Catalysis; Escherichia coli; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Nitrophenols; Penicillin Amidase; Spectrophotometry; Time Factors

HbPresbyterian (beta 108Asn --> Lys, HbP) contains an additional positive charge (per alpha beta dimer) in the middle of the central cavity and exhibits a lower oxygen affinity than wild-type HbA in the presence of chloride. However, very little is known about the molecular origins of its altered functional properties. In this study, we have focused on the beta beta cleft of the Hb tetramer. Recently, we developed an approach for quantifying the ligand binding affinity to the beta-end of the Hb central cavity using fluorescent analogues of the natural allosteric effector 2, 3-diphosphoglycerate (DPG) [Gottfried, D. S., et al. (1997) J. Biol. Chem. 272, 1571-1578]. Time-correlated single-photon counting fluorescence lifetime studies were used to assess the binding of pyrenetetrasulfonate to both HbA and HbP in the deoxy and CO ligation states under acidic and neutral pH conditions. Both the native and mutant proteins bind the probe at a weak binding site and a strong binding site; in all cases, the binding to HbP was stronger than to HbA. The most striking finding was that for HbA the binding affinity varies as follows: deoxy (pH 6.35) > deoxy (pH 7.20) > CO (pH 6.35); however, the binding to HbP is independent of ligation or pH. The mutant oxy protein also hydrolyzes p-nitrophenyl acetate, through a reversible acyl-imidazole pathway linked to the His residues of the beta beta cleft, at a considerably higher rate than does HbA. This implies a perturbation of the microenvironment of these residues at the DPG binding pocket. Structural consequences due to the presence of the new positive charge in the middle of the central cavity have been transmitted to the beta beta cleft of the protein, even in its liganded conformation. This is consistent with a newly described quaternary state (B) for liganded HbPresbyterian and an associated change in the allosteric control mechanism.

Topics: 2,3-Diphosphoglycerate; Asparagine; Binding Sites; Carboxyhemoglobin; Hemoglobin A; Hemoglobins, Abnormal; Humans; Hydrogen-Ion Concentration; Hydrolysis; Lysine; Nitrophenols; Pyrenes; Spectrometry, Fluorescence; Titrimetry

The aim of this study is to investigate species differences in the stereoselective hydrolysis for propranolol ester prodrugs in mammalian intestinal mucosa and Caco-2 cells.. Hydrolase activities for propranolol prodrugs and p-nitrophenylacetate in man (age: 51-71 years), the beagle dog (age: 4 years) and Wistar rat (age: 8 weeks) intestinal mucosa, and also in Caco-2 cells (passage between 60-70) were estimated by determining the rate of production of proparanolol and p-nitrophenol, respectively.. The hydrolase activities for both propranolol prodrugs and p-nitrophenylacetate were in the order of man > rat >> Caco-2 cells > dog for intestinal microsomes, and rat > Caco-2 cells = man > dog for intestinal cytosol. Dog microsomes showed stereoselective hydrolysis for propranolol prodrugs, but not those from human or rat. Interestingly, both subcellular fractions of Caco-2 cells showed remarkable R-enantioselectivity except acetyl propranolol. Enzyme kinetic experiments for each enantiomer of butyryl propranolol in microsomes revealed that dog possesses both low and high affinity hydrolases. Both Km and Vmax values in rat were largest among examined microsomes, while Vmax/Km was largest in man. Finally, it was shown that the carboxylesterases might contribute to the hydrolysis of propranolol prodrug in all species by inhibition experiments.. The hydrolase activities for propranolol prodrugs and p-nitrophenylacetate in intestinal mucosa showed great species differences and those in human intestine were closer to those of rat intestine than dog intestine or Caco-2 cells.

Topics: Aged; Animals; Caco-2 Cells; Cytosol; Dogs; Esters; Humans; Hydrolases; Intestinal Mucosa; Male; Microsomes; Middle Aged; Nitrophenols; Prodrugs; Propranolol; Rats; Rats, Wistar; Species Specificity; Stereoisomerism

Pectinesterase (PE), from commercial orange peels or ammonium sulfate fractionation (50-80% saturation) of pea pods, was detected on polyacrylamide gels after native acidic polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate (SDS)-PAGE by using the synthetic substrate beta-naphthyl acetate (beta-NA). The release of beta-naphthol (at 322 nm) from beta-NA was proportional to PE activity. The PE activity bands on polyacrylamide gels after native acidic PAGE or SDS-PAGE were stained with a combination of tetrazotized o-dianisidine and beta-NA. This fast and sensitive method can be used for enzyme purification and characterization.

Topics: Acrylic Resins; Carboxylic Ester Hydrolases; Coloring Agents; Dianisidine; Electrophoresis, Polyacrylamide Gel; Nitrophenols; Pectins; Pisum sativum; Ruthenium Red; Sodium Dodecyl Sulfate; Staining and Labeling; Substrate Specificity

An intracellular novel lipase which can hydrolyze t-butyl octanoate (TBO) was purified to homogeneity from crude cell-free extracts of Burkholderia (formerly Pseudomonas) sp. YY62 with 9% overall yield. Seventy-four-fold purification was achieved by ammonium-sulfate precipitation, three consecutive open-column chromatographies (DEAE anion-exchange, Sepharose CL-6B gel-filtration, and the second DEAE anion-exchange columns), and two HPLCs (TSK G2000SWXL gel-filtration and phenyl 5PW hydrophobic interaction columns). Enzymes hydrolyzing p-nitrophenyl acetate were separated into two peaks (peak I and II) on the hydrophobic HPLC, and only peak II was found to have TBO-hydrolyzing activity. The peak preparation showed a single band of 40 kDa on SDS-PAGE and a molecular mass of 39 kDa on gel-filtration under non-denatured conditions, indicating the monomeric nature of the TBO-hydrolyzing lipase. The lipase showed maximum activity at pH 7.0 and 28 degrees C. The N-terminal 15 amino acid residues were determined as Met-Asp-Phe-Tyr-Asp-Ala-Asn-Glu-Thr-Arg-His-Pro-Glu-Gln-Arg, which showed no homology to known proteins, suggesting that the purified enzyme may belong to a novel class of hydrolase.

Topics: Amino Acid Sequence; Burkholderia; Caprylates; Chemical Precipitation; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Hot Temperature; Hydrogen-Ion Concentration; Hydrolysis; Lipase; Molecular Sequence Data; Molecular Weight; Nitrophenols; Sequence Analysis

The interaction of native and Co(II)-substituted isozymes I and II of carbonic anhydrase (CA) with histamine, a well-known activator, was investigated kinetically, spectroscopically, and X-ray crystallographically. This activator is of the noncompetitive type with 4-nitrophenyl acetate and CO2 as substrates for both HCA I and HCA II. The electronic spectrum of the adduct of Co(II)-HCA II with histamine is similar to the spectrum of the Co(II)-HCA II-phenol adduct, being only slightly different from that of the uncomplexed enzyme. This is the first spectroscopic evidence that the activator molecule binds within the active site, but not directly to the metal ion. X-ray crystallographic data for the adduct of HCA II with histamine showed that the activator molecule is bound at the entrance of the active site cavity in a position where it may actively participate in shuttling protons between the active site and the bulk solvent. The role of the activators and the reported X-ray crystal structure of the HCA II-histamine adduct has prompted us to reexamine the X-ray structures of the different CA isozymes in order to find a structural basis accounting for their large differences in catalytic rate. A tentative explanation is proposed on the basis of possible pathways of proton transfer, which constitute the rate-limiting step in the catalytic reaction.

Topics: Carbonic Anhydrases; Cobalt; Crystallography, X-Ray; Enzyme Activation; Histamine; Humans; Hydrogen Bonding; Models, Molecular; Nitrophenols; Protein Conformation; Protein Structure, Tertiary; Spectrophotometry; Zinc

A convenient procedure for thiolsubtilisin purification from an admixture of subtilisin involving affinity chromatography on bacitracin-Sepharose is presented. Thiolsubtilisin activity was measured by hydrolysis of p-nitrophenyl acetate, p-nitroanilide-peptide (Glp-Ala-Ala-Leu-pNA), and azocasein. The thiolenzyme catalyzes peptide synthesis. Under these conditions only activated peptide esters, e.g., p-chlorophenyl, N-hydroxysuccinimide, or p-nitrophenyl esters form peptide bonds during interaction with appropriate nucleophiles such as peptides and their derivatives and amino acid amides.

Topics: Amino Acids; Bacillus subtilis; Bacitracin; Caseins; Chromatography, Affinity; Chromatography, Gel; Hydrolysis; Nitrophenols; Peptide Biosynthesis; Peptides; Sepharose; Substrate Specificity; Subtilisins

To explore the role that surface and active center charges play in electrostatic attraction of ligands to the active center gorge of acetylcholinesterase (AChE), and the influence of charge on the reactive orientation of the ligand, we have studied the kinetics of association of cationic and neutral ligands with the active center and peripheral site of AChE. Electrostatic influences were reduced by sequential mutations of six surface anionic residues outside of the active center gorge (Glu-84, Glu-91, Asp-280, Asp-283, Glu-292, and Asp-372) and three residues within the active center gorge (Asp-74 at the rim and Glu-202 and Glu-450 at the base). The peripheral site ligand, fasciculin 2 (FAS2), a peptide of 6.5 kDa with a net charge of +4, shows a marked enhancement of rate of association with reduction in ionic strength, and this ionic strength dependence can be markedly reduced by progressive neutralization of surface and active center gorge anionic residues. By contrast, neutralization of surface residues only has a modest influence on the rate of cationic m-trimethylammoniotrifluoroacetophenone (TFK+) association with the active serine, whereas neutralization of residues in the active center gorge has a marked influence on the rate but with little change in the ionic strength dependence. Brownian dynamics calculations for approach of a small cationic ligand to the entrance of the gorge show the influence of individual charges to be in quantitative accord with that found for the surface residues. Anionic residues in the gorge may help to orient the ligand for reaction or to trap the ligand. Bound FAS2 on AChE not only reduces the rate of TFK+ reaction with the active center but inverts the ionic strength dependence for the cationic TFK+ association with AChE. Hence it appears that TFK+ must traverse an electrostatic barrier at the gorge entry imparted by the bound FAS2 with its net charge of +4.

Topics: Acetophenones; Acetylcholinesterase; Acetylthiocholine; Animals; Binding Sites; Cations; Cholinesterase Inhibitors; Elapid Venoms; Kinetics; Ligands; Mice; Models, Molecular; Mutagenesis, Site-Directed; Nitrophenols; Osmolar Concentration; Protein Conformation; Static Electricity; Thermodynamics

The lung is highly susceptible to ethyl carbamate (EC)-induced tumorigenesis. Our goal in this study was to investigate the in vitro isozyme-selective metabolism of EC in lung microsomes by cytochrome P450 and carboxylesterase enzymes. Our results showed that incubations with EC produced significant reduction in p-nitrophenol (PNP) hydroxylation and N-nitrosodimethylamine (NDMA) demethylation; there were no alterations in 7-pentoxyresorufin- and 7-ethoxyresorufin O-dealkylase activities. Reaction of microsomes with an inhibitory CYP2E1 antibody and subsequent reaction with EC abolished the EC-induced diminution in NDMA demethylase activity. Carboxylesterase activity, as assessed by hydrolysis of p-nitrophenyl acetate, was significantly decreased in microsomes incubated with EC. Reactions with EC in conjunction with the carboxylesterase inhibitors, paraoxon (PAX) or phenylmethylsulfonyl fluoride (PMSF), abolished the EC-induced decrease in carboxylesterase activity; PAX is a broad-spectrum carboxylesterase inhibitor, whereas PMSF is a specific inhibitor of hydrolase A, a carboxylesterase isozyme. Incubations of EC in combination with either PAX or PMSF exacerbated the EC-induced reduction in PNP hydroxylase and NDMA demethylase activities. Alterations in immunodetectable CYP2E1 protein levels were not apparent in microsomes incubated with EC alone, but the amounts were decreased in reactions with EC in conjunction with either PAX or PMSF. Immunoblotting with antibodies for the carboxylesterase isozymes, hydrolase A and B, revealed loss of immunodetectable hydrolase A in microsomes incubated with EC, PAX, or PMSF. However, immunodetectable hydrolase B was only decreased in microsomes reacted with PAX but not with PMSF or EC. These findings correlated with our covalent binding data, which showed that levels of binding of [14C-ethyl]EC to lung microsomes were significantly higher in incubations conducted in conjunction with PAX or PMSF, compared with control levels. Antibody inhibition of the CYP2E1 enzyme significantly reduced the extent of binding. Our results demonstrated that EC metabolism in lung microsomes, as estimated from magnitudes of covalent binding, is mediated by the P450 isozyme CYP2E1 and the carboxylesterase isozyme hydrolase A.

Topics: Animals; Binding, Competitive; Biotransformation; Carboxylesterase; Carboxylic Ester Hydrolases; Carcinogens; Cholinesterase Inhibitors; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP2B1; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme System; Female; Hydrolysis; Hydroxylation; Immunoblotting; Inactivation, Metabolic; Isoenzymes; Lung; Mice; Microsomes; Nitrophenols; Paraoxon; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Urethane

Vector control programmes in many countries face the dual problems of parasite drug resistance and insecticide resistance in the insect vectors of the disease. Here we report for the first time a new esterase-based insecticide resistance mechanism in the filariasis vector Culex quinquefasciatus. The field collected COL strain of C. quinquefasciatus from Columbia was heterogeneous for organophosphorus insecticide resistance. On native polyacrylamide gels it had an elevated beta-naphthyl acetate specific esterase with the same Rf as that for the Est beta 1s involved in insecticide resistance in other strains of this mosquito species. After five generations of temephos insecticide selection, both the esterase specific activity with p-nitrophenyl acetate and the temephos LC50 values were increased, suggesting that elevation of esterase activity was the underlying mechanism of resistance. Western blots with antisera raised to Est alpha 2(1) and Est beta 2(1) from C. quinquefasciatus indicated that the COL strain had an elevated Est alpha 3 enzyme which co-migrated on native gels with Est beta 1. Southern blots indicated that an est alpha 3 gene was amplified in the COL strain and a Cuban mosquito strain (MRes), although the restriction digest patterns of the est beta 1 genes in these two strains are different. In contrast, the Californian TEMR strain, with the amplified est beta 1(1) gene, had no associated elevated Est alpha. Restriction digest patterns for COL and TEMR DNA suggest that they contain an identical est beta 1(1) gene, but our data suggest that the est alpha 3 gene occurs on the same amplicon as an est beta 1 gene although the genes are probably > 10 kb apart. Hence, either the COL strain has two est beta 1 genes or the est beta 1(1) amplicon in TEMR has been disrupted at some stage during the long colonisation of this strain and the amplified est alpha has been lost.

Topics: Animals; Culex; Esterases; Filariasis; Insect Vectors; Insecticide Resistance; Insecticides; Nitrophenols; Temefos

The catalytic zinc ion of human carbonic anhydrase II (CAII) is coordinated by three histidine ligands (H94, H96, and H119) and a hydroxide ion with tetrahedral geometry. Structural and functional analysis of variants in which the zinc ligands H94 and H119 are substituted with asparagine and glutamine, and comparison with results obtained with aspartate and glutamate substitutions indicate that the neutral ligand field provided by the protein optimizes the electrostatic environment for the catalytic function of the metal ion, including stabilization of bound anions. This is demonstrated by catalytic activity measurements for ester hydrolysis and CO2 hydration, as well as sulfonamide inhibitor affinity assays. High-resolution X-ray crystal structure determinations of H94N, H119N, and H119Q CAIIs reveal that the engineered carboxamide side chains coordinate to zinc with optimal stereochemistry. However, zinc coordination geometry remains tetrahedral only in H119Q CAII. Metal geometry changes to trigonal bipyramidal in H119N CAII due to the addition of a second water molecule to the zinc coordination polyhedron and also in H94N CAII due to the displacement of zinc-bound hydroxide by the bidentate coordination of a Tris molecule. Possibly, the bulky histidine imidazole ligands of the native enzyme play a role in disfavoring trigonal bipyramidal coordination geometry for zinc. Protein-metal affinity is significantly compromised by all histidine --> carboxamide ligand substitutions. Diminished affinity may result from significant movements (up to 1 A) of the metal ion from its position in the wild-type enzyme, as well as the associated, minor conformational changes of metal ligands and their neighboring residues.

Topics: Binding Sites; Carbon Dioxide; Carbonic Anhydrases; Catalysis; Crystallography, X-Ray; Enzyme Inhibitors; Esterases; Histidine; Humans; Hydrogen Bonding; Hydrogen-Ion Concentration; Kinetics; Ligands; Models, Molecular; Mutagenesis, Site-Directed; Nitrophenols; Sulfonamides; Zinc

Mammalian albumins have two main structurally selective ligand binding sites. Site I binds drugs such as azapropazone, phenylbutazone and warfarin; whereas benzodiazepines, some dansyl amino acids, such as dansylsarcosine, and short chain fatty acids like octanoic acid interact with site II. However, it is not known if non-mammalian albumins have similar binding loci. In this study, drug binding sites on chicken albumin were investigated using site selective fluorescent probes (warfarin and dansylsarcosine) and p-nitrophenyl acetate (NPA); the hydrolysis of which is selectively inhibited by site II ligands. Azapropazone and phenylbutazone decreased the binding of warfarin and dansylsarcosine to a similar extent. Diazepam and octanoic acid also inhibited binding of the two fluorescent probes in a non-selective manner. However, the fluorescence intensity of the warfarin-chicken albumin complex decreased when the pH was increased from 6.0-9.0; but by contrast, the fluorescence of bound dansylsarcosine remained unchanged. Furthermore, the hydrolysis of NPA was selectively inhibited by dansylsarcosine, diazepam and octanoic acid (ligands selective for site II on mammalian albumins), but not by site I selective ligands such as azapropazone and warfarin. Overall, the results suggest that chicken albumin, like mammalian albumins, has discrete binding sites for warfarin and dansylsarcosine.

Topics: Albumins; Animals; Anti-Inflammatory Agents, Non-Steroidal; Anticoagulants; Apazone; Binding Sites; Chickens; Dansyl Compounds; Humans; Hydrogen-Ion Concentration; Hydrolysis; Ligands; Nitrophenols; Sarcosine; Species Specificity; Warfarin

The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1.s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1.s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis.

Topics: Acylation; Catalysis; Enterobacteriaceae; Hydrolysis; Kinetics; Mutation; Nitrophenols; Penicillin Amidase; Penicillin G; Phenylacetates; Phenylpropionates; Spectrophotometry

Kinetics analysis of the growth of Fusarium sporotrichioides T-424 at 15 degrees C and 25 degrees C in liquid culture for 35 days revealed that production of deacetylated trichothecenes was associated with an increased activity in fungal esterases. High temperature (25 degrees C) favored enzyme production and enhanced esterase activity. Electrophoresis of crude extracts from the mycelia of F. sporotrichioides T-424 with carboxylesterase staining revealed that several esterases were produced by the fungus. Four carboxylesterase isoenzymes (I-IV) were separated on a DEAE-Sephadex anion exchange column. Type (III) esterase, having activities with the substrate 4-nitrophenylacetate and acetanilide, as well as hydrolytic activity for T-2 toxin and acetyl-T-2 toxin, was partially purified with ammonium sulfate precipitation, immunoaffinity column chromatography, and DEAE-Sephadex A-50 chromatography. The esterase (III) had a molecular weight around 68 kDa in SDS-PAGE. For the deacylation of T-2 toxin and acetyl-T-2 toxin, type (III) esterase had a high specificity for the acetyl group at the C-3 and C-4 positions. The Km values for acetyl-T-2 and T-2 toxin were found to be 41.35 microM and 0.38 microM, respectively. The Km value for the acetyl group at C-3 is 110 times greater than for that at C-4.

Topics: Acetanilides; Ammonium Sulfate; Carboxylesterase; Carboxylic Ester Hydrolases; Chromatography, Affinity; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Culture Media; Electrophoresis, Polyacrylamide Gel; Esterases; Fusarium; Hydrogen-Ion Concentration; Hydrolysis; Isoenzymes; Kinetics; Molecular Weight; Nitrophenols; Temperature; Trichothecenes

We report the design of a cyclic, eight-residue peptide that possesses the catalytic triad residues of the serine proteases. A manually built model has been relaxed by 0.3 ns of molecular dynamics simulation at room temperature, during which no major changes occurred in the peptide. The molecule has been synthesised and purified. Two-dimensional NMR spectroscopy provided 35 distance and 7 torsion angle constraints, which were used to determine the three-dimensional structure. The experimental conformation agrees with the predicted one at the beta-turn, but deviates in the arrangement of the disulphide bridge that closes the backbone to a ring. A 1.2 ns simulation at 600 K provided extended sampling of conformation space. The disulphide bridge reoriented into the experimental arrangement, producing a minimum backbone rmsd from the experimental conformation of 0.8 A. At a later stage in the simulation, a transition at Ser3 produced more pronounced high-temperature behaviour. The peptide hydrolyses p-nitrophenyl acetate about nine times faster than free histidine.

Topics: Amino Acid Sequence; Computer Simulation; Hydrolysis; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Molecular Structure; Nitrophenols; Peptides, Cyclic; Protein Conformation; Serine Endopeptidases; Thermodynamics

A polyallylamine carrying long hydrophobic dodecyl groups and adenine residues as side chains (PALAD C12) may be able to catalyze the hydrolysis of N-carbobenzoxy-l-alanine p-nitrophenyl ester (N-Cbz-Ala) as well as p-nitrophenyl acetate (pNPA). The progress curve of hydrolysis of the former displays a long lag and apparently no steady state. After this transient the rate falls off due to the accumulation of the products. Conversely, the hydrolysis of p-nitrophenyl acetate displays classical burst kinetics followed by a slow decline of the reaction rate.Theoretical considerations show that a steady state may be expected to occur only if the concentration of the free catalyst is very small during the reaction. This condition is sufficient to allow the rate of disappearance of the substrate to be equal to the rate of appearance of the products, which is precisely a condition for the existence of a steady state. If the catalyst is poorly active and has a loose affinity for its substrate and product, the measurement of a significant reaction rate will require a much larger concentration of the catalyst. Therefore, under these conditions, one cannot expect a steady state to occur. The mathematical expression of the error made in the steady-state assumption has been derived. This error increases with the catalyst concentration and decreases if the affinity of the substrate for the catalyst is high. Therefore the lack of steady state is associated with the affinity (or the dissociation) of the substrate and the product for the catalyst. When this affinity is low, the free concentration of the catalyst during the reaction is high and one cannot expect a steady state to occur. This is precisely what takes place with N-Cbz-Ala.A mathematical expression of the rate of hydrolysis of N-Cbz-Ala and of any reactant that displays this type of kinetics may be derived at the end of the transient when the rate is close to its maximum value. Under these conditions the rate cannot follow classical Michaelis-Menten kinetics and displays positive cooperativity. It may therefore be speculated that primordial template-like catalysts that were displaying a poor affinity for their substrates and products were already exhibiting apparent positive cooperativity in the kinetic reactions they were able to catalyze.

Topics: Alanine; Catalysis; Evolution, Chemical; Hydrolysis; Kinetics; Models, Chemical; Nitrophenols; Polyamines; Purines

The interfacial activation of Candida antarctica lipase A (CALA) and B (CALB) has been investigated and compared with that of Humicola lanuginosa lipase (HLL). CALB displayed no interfacial activation towards p-nitrophenyl butyrate (PNPB) when exceeding the solubility limit of the substrate. No activation was observed towards p-nitrophenyl acetate (PNPA) at the addition of sodium dodecyl sulfate (SDS) nor in the presence of a solid polystyrene surface. The catalytic action of CALB was very different from that of Humicola lanuginosa lipase, which showed a pronounced interfacial activation with the same substrates. The basis for the anomalous behaviour of CALB is proposed to be due to the absence of a lid that regulates the access to the active site. In contrast to CALB, CALA expressed interfacial activation, but the activation was not as prominent as for Humicola lanuginosa lipase (HLL). The structural basis for the activation of CALA is unknown.

Topics: Adsorption; Binding Sites; Butyrates; Candida; Enzyme Activation; Lipase; Mitosporic Fungi; Nitrophenols; Protein Conformation; Protein Structure, Secondary; Sodium Dodecyl Sulfate; Surface Properties; Triglycerides

In this paper we describe a fast and mild method based on the use of a unique cation exchanger and buffers containing ethylene glycol and salt for the purification of the myelin basic protein (MBP; MW 18.5 kDa). MBP thus purified hydrolyses catalytically p-nitrophenyl acetate. This esterase activity facilitates not only the purification of MBP but also indicates that probably it is in its native state, i.e. there is a good chance that the purified molecules are structurally and chemically identical. This is a prerequisite to obtain crystals appropriate for x-ray diffraction and other studies.

Topics: 4-Nitrophenylphosphatase; Amino Acid Sequence; Animals; Cattle; Chromatography, Ion Exchange; Detergents; Electrophoresis, Polyacrylamide Gel; Esterases; Ethylene Glycol; Ethylene Glycols; Female; Humans; Kinetics; Molecular Sequence Data; Molecular Weight; Myelin Basic Protein; Nitrophenols; Rabbits; Sequence Homology, Amino Acid; Sodium Chloride; Spinal Cord

The binding of some cephalosporins to human serum albumin was studied using probes for the so-called I, II, bilirubin and fatty acids binding sites. The results showed that cephradine and cefsulodin bind to site II, cefaclor, cefamandole, cefsulodin, cephaloglycin and cefadroxil bind to the bilirubin binding site, while cefaclor does it to the fatty acid binding site. No binding of these cephalosporins to site I of albumin was found. The binding produced a perturbation on the N-B equilibrium of albumin, stabilizing the N conformational form, which suggests that the N form of albumin has more affinity with the cephalosporins than the B form. This finding gives support to the assumption that the binding of cephalosporins to site II, bilirubin and fatty acids binding sites affects the N-B transition of albumin.

Topics: Bilirubin; Binding Sites; Binding, Competitive; Cephalosporins; Dansyl Compounds; Fatty Acids; Fluorescence; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Molecular Structure; Nitrophenols; Protein Binding; Serum Albumin

Placental biotransformation reactions may modulate the effect a xenobiotic has on the developing fetus. However, in spite of the critical role the placenta plays in supporting fetal life, little is known about the pharmacology and toxicology of the human placenta. Our laboratory has previously characterized the N-acetylation activity of the human term placenta. This activity is predominantly attributable to the NAT1 form of arylamine N-acetyltransferase (NAT). Although acetylation is generally thought to be a detoxifying reaction, both N-acetylation and deacetylation reactions play an important role in the activation of carcinogenic arylamines to their reactive and toxic forms. In the current study we characterized the activity of human placental NAT and deacetylase toward the carcinogenic arylamine, 2-aminofluorene (AF) and its acetylated metabolite, 2-acetylaminofluorene (AAF). 2-Aminofluorene is a synthetic, prototype carcinogenic arylamine compound, and its metabolism has been extensively studied in the laboratory. Our data show that the affinity (Km = 24.2 +/- 1.66 mumol/L; mean +/- SEM, n = 6) and maximal velocity (Vmax = 4.29 +/- 0.33 nmol/min/mg; mean +/- SEM, n = 6) of AF N-acetylation by human placenta are similar to those in human liver. The deacetylation of AAF to AF by placental microsomes may be catalyzed by a carboxylesterase. However, our studies with inhibitors reveal that the characteristics of human placental deacetylation activity differ from that of human liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2-Acetylaminofluorene; Acetylation; Biotransformation; Carcinogens; Female; Fluorenes; Humans; Isocyanates; Microsomes, Liver; Nitrophenols; Placenta; Pregnancy

Seven highly active inhibitors against carbonic anhydrase (CA, EC 4.2.1.1), punicalin (2), punicalagin (3), granatin B (5), gallagyldilactone (7), casuarinin (8), pedunculagin (9) and tellimagrandin I (10), and four weakly active inhibitors, gallic acid (1), granatin A (4), corilagin (6) and ellagic acid (11), were isolated from the pericarps of Punica granatum L. (Punicaceae). They are ellagitannins. The type of inhibition by 3 and 7 using p-nitrophenyl acetate as a substrate, is noncompetitive. The structure-activity relationship of inhibitory effects on CA is discussed.

Topics: Animals; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Cattle; Hydrolyzable Tannins; Kinetics; Nitrophenols; Plant Extracts; Plants; Structure-Activity Relationship; Tannins

1. Differences in the ability of metabolically-inert peroxisome proliferators (perfluoro-n-decanoic acid (PFDA, C10), perfluoro-n-octanoic acid (PFOA, C8), perfluorooctane sulphonic acid (PFOS, C8) and 1-H,1-H-pentadecafluoro-n-octanol (PFOL, C8)) to induce three forms of hepatic microsomal carboxylesterase, namely RL1, RL2 and RH1, in the male rat were studied by measuring changes in hydrolytic activities towards p-nitrophenyl acetate (PNPA), isocarboxazid (ISOC) and butanilicaine (BUTA), which are thought to be specific substrates for RL1, RL2 and RH1, respectively, and by evaluating changes in the contents of the three isozymes by radial immunodiffusion assay with specific antibodies. 2. The administration of PFDA rather specifically decreases PNPA hydrolase activity and RL1 content. On the other hand, PFOA, PFOS and PFOL markedly increase all three hydrolase activities and the content of all three isozymes (except RH1 in the case of PFOA, where the increase was not statistically significant). 3. The correlations between hydrolase activities and isozyme contents supported specificity of the three substrates, with the exception that the content of the predominant isozyme, RL2, showed a higher correlation with BUTA hydrolase activity than with ISOC hydrolase activity. 4. In conclusion, we have demonstrated that metabolically-inert perfluorinated fatty acids induce hepatic microsomal carboxylesterase isozymes, as determined by radial immunodiffusion analysis using specific antibodies. This is the first report that perfluorinated fatty acid affect carboxylesterase isozymes in rat liver microsomes, and is indicative of the importance of peroxisome proliferators in hepatic metabolism of xenobiotics. Further work is needed to determine the regulatory mechanisms involved.

Topics: Acetanilides; Animals; Carboxylic Ester Hydrolases; Enzyme Induction; Fatty Acids; Fluorocarbons; Isocarboxazid; Isoenzymes; Male; Microsomes, Liver; Nitrophenols; Rats; Rats, Sprague-Dawley

A novel membrane-bound esterase was purified from mouse hepatic microsomes. The purified protein (ES46.5K) showed a single protein-staining band on sodium dodecyl sulfate-polyacrylamide gel with a minimum molecular weight of 46.5 Kdalton. ES46.5K possessed esterase activity toward 11-acetoxy-delta 8-tetrahydrocannabinol (11-OAc-delta 8-THC) (27.1 mumol/min/mg protein) and p-nitrophenylacetate (119 mumol/min/mg protein), and these activities were 38 and 47 times, respectively, as high as those of microsomes. The N-terminal amino acid sequence of the protein was as follows: G-K-T-I-S-L-L-I-S-V-V-L-V-A-Y-Y-L-Y-I. This sequence has no homology to those of the known carboxylesterases, indicating that this enzyme is a novel type of esterase bound to the microsomal membrane.

Topics: Amino Acid Sequence; Animals; Binding Sites; Dronabinol; Electrophoresis, Polyacrylamide Gel; Esterases; Male; Mice; Microsomes, Liver; Molecular Sequence Data; Molecular Weight; Nitrophenols

The hydrolase activities of bovine carbonic anhydrase B (BCA II carbonate hydrolyase, EC 4.2.1.1) were modified by cyanogen (C2N2, N identical to C-C identical to N, ethanedinitrile) with decreases in Vmax of as much as 99%. This was not accompanied by a reduction in hydrolyase activity. These changes were not reversed at lower pH values but the enzymatic activity was restored by incubation at pH 10. 14C-labeled glycine ethyl ester ([14C]GEE) specifically and covalently bound to the cyanogen-treated BCA II, as verified by HPLC and 14C monitoring. It was shown that sites of cyanogen-introduced modifications in BCA II could be effectively labeled and identified by incubation with the nucleophile [14C]GEE. Three radiolabeled tryptic peptides from BCA II arising from a labeling process designed to study cyanogen-induced modifications leading to nucleophile labile covalent bonds have been isolated. The residues identified by [14C]GEE labeling were Asp-34, Glu-117 and Asp-152. Three moieties attached to the omega-carboxyls by C2N2 were tentatively identified by molecular modeling; they were Arg-111, His-107 and/or His-119 and Ser-216, respectively. The use of C2N2 afforded a means to compare the salt bridges in two species and showed that two of three were not conserved.

Topics: Amino Acids; Animals; Binding Sites; Carbonic Anhydrases; Cattle; Chromatography, High Pressure Liquid; Glycine; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Nitriles; Nitrophenols; Trypsin

The catalytic mechanism of carbonic anhydrase includes the reaction of a zinc-bound hydroxide ion with the CO2 substrate. This hydroxide ion is part of a hydrogen-bonded network involving the conserved amino acid residues Thr199, Glu106 and Tyr7. To investigate the functional importance of these residues, a number of site-specific mutants have been made. Thus, Thr199 has been changed to Ala, Glu106 to Ala, Gln and Asp, and Tyr7 to Phe. The effects of these mutations on catalyzed CO2 hydration and ester hydrolysis have been measured, as well as the binding of some inhibitors. The results show that the CO2 hydration activity of the mutant with Phe7 is only marginally reduced, whereas the esterase activity is larger than that of unmodified enzyme. It is concluded that Tyr7 is not a functionally required element of the hydrogen-bonded network. The CO2 hydration activity (kcat as well as kcat/Km) and the esterase activity of the mutant with Ala199 are reduced about 100-fold. The affinity for the sulfonamide inhibitor, dansylamide, is only slightly reduced while the mutant has an enhanced affinity for bicarbonate and the anionic inhibitor, SCN-. The activities of the mutants with Ala106 and Gln106 are also reduced. The reduction of the esterase activity is about 100-fold, while kcat for CO2 hydration has decreased by a factor of 1000. The parameter kcat/Km is only about one order of magnitude smaller for these mutants than for the unmodified enzyme. The binding of dansylamide and another sulfonamide inhibitor, acetazolamide, are about 20-times weaker to the mutant with Gln106 than to unmodified enzyme. These results suggest important roles for Thr199 and Glu106 in carbonic anhydrase catalysis. The mutant with Asp106 is almost fully active suggesting that the structure has undergone a compensatory change to maintain the interaction between residue 106 and Thr199.

Topics: Amino Acid Sequence; Binding Sites; Carbon Dioxide; Carbonic Anhydrases; Catalysis; Conserved Sequence; Dansyl Compounds; Glutamine; Humans; Hydrogen Bonding; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Mutagenesis, Site-Directed; Nitrophenols; Spectrometry, Fluorescence; Structure-Activity Relationship; Threonine; Tyrosine

Purified human milk lipoamidase was digested with endoproteinase Lys-C and the digested peptides were subjected to gasphase microsequence analysis. The sequencing of three isolated peptides of human milk lipoamidase revealed the identity of this protein with human milk bile salt-stimulated lipase (pancreatic cholesterol esterase). The identity of the cholesterol esterase with lipoamidase was confirmed by expressing a recombinant form of rat pancreatic cholesterol esterase and testing for lipoamidase activity of the recombinant protein. The results showed that the recombinant cholesterol esterase displayed both lipolytic and lipoamidase activities and was capable of hydrolysing triacetin and lipoyl-4-aminobenzoate (LPAB). The mechanisms of the esterase and amidase activities of the enzyme were further tested by determining enzyme activity in a mutagenized cholesterol esterase with a His435-->Gln435 substitution. This mutation has been shown previously to abolish enzyme activity against esterase substrates [DiPersio, Fontaine and Hui (1991) J. Biol. Chem. 266, 4033-4036]. We showed that the mutagenized protein was effective in hydrolysing the amidase substrate LPAB and displayed similar enzyme kinetics to those of the native enzyme. These data indicate that the mechanism for the cholesterol esterase hydrolysis of lipoamides is different from that of the hydrolysis of substrates with an ester linkage. The presence of an enzyme in the gastrointestinal tract capable of both ester and amide hydrolysis suggests an important role for this protein in the digestion and absorption processes.

Topics: 4-Aminobenzoic Acid; Amidohydrolases; Amino Acid Sequence; Bile Acids and Salts; Female; Humans; Hydrolysis; Metalloendopeptidases; Milk, Human; Molecular Sequence Data; Mutagenesis; Nitrophenols; Pancreas; para-Aminobenzoates; Peptide Fragments; Recombinant Proteins; Sterol Esterase; Thioctic Acid; Triacetin

1. The kinetics and inhibition of p-nitrophenylacetate hydrolysis by cytosolic esterases of 1-day old female honey bees, Apis mellifera L., were studied. 2. The calculated values obtained were Km = 2.27 x 10(-5)M and Vmax = 2.48 x 10(-8) mol/s per mg protein. 3. The inhibition mechanisms examined for four organophosphorus insecticides were highly competitive in nature and based on competitive inhibition coefficients the order of toxicity was naled > dichlorvos > cis-mevinphos = trans-mevinphos. 4. Comparisons are made with the alfalfa leafcutting bee, Megachile rotundata (Fab).

Topics: Animals; Bees; Cytosol; Enzyme Activation; Esterases; Female; Honey; Hydrolysis; Inactivation, Metabolic; Insecticides; Kinetics; Medicago sativa; Nitrophenols; Organophosphorus Compounds

Topics: Animals; Dinitrochlorobenzene; Glutathione Transferase; Indicators and Reagents; Liver; Nitrophenols; Oxazines; Rats; Rats, Wistar; Substrate Specificity; Triazenes; Xanthenes

The fluorescent probes warfarin and dansylsarcosine are known to selectively interact with binding sites I and II, respectively, on human albumin. This paper investigates whether similar binding sites exist on bovine, dog, horse, sheep and rat albumins. Binding sites on albumins were studied by: (1) displacement of warfarin and dansylsarcosine by site I (phenylbutazone) and site II (diazepam) selective ligands; (2) the effects of non-esterified fatty acids (carbon chain lengths: C5-C20) and changes in pH (6-9) on the fluorescence of warfarin and dansylsarcosine; and (3) the ability of site selective ligands to inhibit hydrolysis of 4-nitrophenyl acetate. For bovine, dog, horse, human and sheep albumins the fluorescence of bound warfarin and dansylsarcosine was selectively decreased by phenylbutazone and diazepam, respectively. For these albumins medium chain fatty acids (C1-C12) reduced the fluorescence of dansylsarcosine (maximum inhibition with C9) whereas long chain acids (C12-C20) enhanced the fluorescence of warfarin (maximum increases with C12). In addition, changes in pH from 6 to 9 increased the fluorescence of warfarin and although site I ligands (warfarin/phenylbutazone) had no pronounced effects on 4-nitrophenyl acetate hydrolysis, site II ligands (dansylsarcosine/diazepam) significantly inhibited this reaction. Rat albumin behaved differently from the other albumins studied in that the C12-C20 fatty acids and changes in pH did not enhance the fluorescence of warfarin. Moreover, the differential effects of site I and site II ligands on the fluorescence of warfarin/dansylsarcosine and hydrolysis of 4-nitrophenyl acetate were less apparent with rat albumin. The results suggest bovine, dog, horse and sheep albumins have binding sites for warfarin and dansylsarcosine with similar properties to sites I and II on human albumin. By contrast, the warfarin binding site and to a lesser degree the dansylsarcosine site, of rat albumin have different characteristics from these sites on the other albumins studied.

Topics: Albumins; Animals; Binding Sites; Binding, Competitive; Cattle; Dansyl Compounds; Diazepam; Dogs; Fatty Acids, Nonesterified; Fluorescence; Horses; Humans; Hydrogen-Ion Concentration; Kinetics; Nitrophenols; Phenylbutazone; Rats; Sarcosine; Sheep; Warfarin

The in situ modification of an immobilized human serum albumin (HSA) high-performance liquid chromatographic chiral stationary phase by p-nitrophenyl acetate is reported. This procedure, which is thought to affect primarily a single reactive tyrosine residue within the protein structure, influenced the chromatographic retention and enantioselectivity factors of a wide range of solutes. For certain solutes, increases in both capacity factor and chiral resolution were observed. Ultrafiltration studies on representative test solutes using free HSA, treated in a similar manner to the immobilized protein, gave similar results as the chromatographic observations, indicating that the latter effects are not artifactual results of immobilization. The effect of the modification of HSA on the binding behavior of drugs reportedly sharing the site predominantly affected by the derivatization, namely, the indole-benzodiazepine binding site, varied greatly. This observation suggests that the affected binding area is not a single, tightly structurally defined site.

Topics: Acetylation; Chromatography, High Pressure Liquid; Humans; In Vitro Techniques; Nitrophenols; Protein Binding; Serum Albumin

A new method for phenotyping human serum arylesterase (EC 3.1.1.2) is described and evaluated. The aromatic esters, phenyl acetate and 4-nitrophenyl acetate, were compared as substrates for spectrophotometric measurement of arylesterase activity. A method for arylesterase phenotyping, based upon inhibition of the enzymatic hydrolysis of 4-nitrophenyl acetate by phenyl acetate, was developed. The method was applied to serum samples from 158 blood donors and showed a distinct separation of the three phenotypes defined by a reference method based on the ratio of paraoxonase activity to arylesterase activity using paraoxon and phenyl acetate as substrates. The method was adapted to a Cobas-Fara centrifugal analyser.

Topics: Alleles; Blood Donors; Carboxylic Ester Hydrolases; Humans; Hydrolysis; Isoenzymes; Kinetics; Nitrophenols; Phenotype; Phenylacetates; Substrate Specificity

Modification of a single arginine residue per subunit of rabbit muscle apo-D-Glyceraldehyde-3-phosphate dehydrogenase does not affect the rate of hydrolysis of p-nitrophenyl acetate catalyzed by the enzyme, but locks the tetramer in a conformation wherein only two active sites are functioning. The modified enzyme also exhibits half-of-the sites reactivity towards iodoacetate and iodoacetamide. On the other hand, its NAD(+)-binding characteristics remain unchanged. Evidence is presented supporting the view that mechanisms of half-of-the-sites reactivity and negative cooperativity in coenzyme binding are different. The results are consistent with a built-in asymmetry of the tetramer and suggest that the arginine residue (probably Arg-231) controls the conformational transition between the asymmetric and symmetric states of the tetramer.

Topics: Animals; Apoenzymes; Arginine; Binding Sites; Glyceraldehyde-3-Phosphate Dehydrogenases; Hydrolysis; Iodoacetamide; Iodoacetates; Iodoacetic Acid; Kinetics; Macromolecular Substances; Muscles; NAD; Nitrophenols; Protein Conformation; Rabbits; Structure-Activity Relationship

4-trans-(NN-Dimethylamino)cinnamaldehyde (an aldehyde, DACA) and 4-trans-(NN-dimethylamino)cinnamoylimidazole (an amide, DACI) have been shown to be substrates for human aldehyde dehydrogenase (EC 1.2.1.3) which form chromophoric covalent intermediates. The spectra of covalent intermediates from both the cytoplasmic (E1) and mitochondrial (E2) isoenzymes derived from DACA and DACI were compared. The spectra were similar when either substrate was used, and also when the two isoenzymes were compared, and resembled that obtained for 4-trnas-(NN-dimethylamino)cinnamoyl-N-acetylcysteine, but differed from the spectrum of 4-trans-(NN-dimethylamino)cinnamoyl ethyl ester. After extensive digestion of the covalent intermediates from both 3H-labelled DACA and DACI with Pronase and purification, the labelled amino acid was identified as cysteine. Covalent intermediates from both DACA and DACI were also digested with trypsin, and labelled peptides were purified by ion-exchange and reverse-phase chromatography. Amino acid sequence analysis showed that the peptide comprising residues 273-307 was labelled by both DACA and DACI. The radioactive label at cysteine residues 301-303 of the primary structure could be unequivocally identified by employing the DACA derivative. Assignment of label to cysteine-302 was achieved by employing iodoacetamide-labelled E1 isoenzyme (iodoacetamide specifically labels cysteine-302), in which case there was no formation of the covalent intermediate from either DACA or DACI. In addition, cysteine-302 is the only cysteine residue conserved in all aldehyde dehydrogenases sequenced. Thus cysteine-302 is the amino acid residue that forms a covalent intermediate with both aldehyde and ester substrates.

Topics: Alcohol Dehydrogenase; Aldehydes; Amino Acid Sequence; Catalysis; Chloral Hydrate; Chromatography, Liquid; Cinnamates; Esters; Humans; Hydrolysis; Imidazoles; Isoenzymes; Kinetics; Molecular Sequence Data; NAD; Nitrophenols; Substrate Specificity

The complete sequence of the horse pancreatic lipase was elucidated by combining polypeptide chain and cDNA sequencing. Among the structural features of horse lipase, it is worth mentioning that Lys373 is not conserved. This residue, which is present in human, porcine and canine lipases, has been assumed to be involved in p-nitrophenyl acetate hydrolysis by pancreatic lipases. Kinetic investigation of the p-nitrophenyl acetate hydrolysis by the various pancreatic lipases and by the C-terminal domain (336-449) of human lipase reveals that this hydrolysis is the result of the superimposition of independent events; a specific linear hydrolysis occurring at the active site of lipase, a fast acylation depending on the presence of Lys373 and a non-specific hydrolysis most likely occurring in the C-terminal domain of the enzyme. This finding definitely proves that pancreatic lipase bears only one active site and raises the question of a covalent catalysis by pancreatic lipases. Moreover, based on sequence comparison with the above-mentioned pancreatic lipases, three residues located in the C-terminal domain, Lys349, Lys398 and Lys419, are proposed as possible candidates for lipase/colipase binding.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA; Dogs; Horses; Humans; Hydrolysis; Kinetics; Lipase; Molecular Sequence Data; Nitrophenols; Pancreas; Pancreatic Juice; Sequence Homology, Nucleic Acid; Swine

1. Homogenates of tissues from females of the nematode Heterodera glycines were clarified by centrifugation and used to initiate characterization of soluble esterases using p-nitrophenyl acetate as the substrate. 2. Optimum temperature and pH were 40 degrees C and 7.2 respectively. 3. Acetazolamide (a carbonic anhydrase inhibitor) at 10(-3) M did not inhibit enzyme activity, indicating that carbonic anhydrase was not present. 4. Phenamiphos (an organophosphate) at 10(-6) M reduced activity by 38%, whereas eserine hemisulfate (a cholinesterase inhibitor) and aldicarb (a carbamate) were not inhibitory at that concentration, indicating that there was no cholinesterase activity. 5. Eserine hemisulfate, aldicarb, and phenamiphos inhibited enzyme activity by 50% (I50) at 5 x 10(-3) M, 7.5 x 10(-4) M, and 6 x 10(-6) M, respectively. 6. Approximately 25% of the activity detected appeared due to A- and/or C-esterases. 7. The data demonstrated that aldicarb and phenamiphos were active against esterases other than acetylcholinesterase.

Topics: Acetazolamide; Aldicarb; Animals; Esterases; Female; Hydrogen-Ion Concentration; Hydrolysis; Insecticides; Nematoda; Nitrophenols; Organophosphorus Compounds; Physostigmine; Temperature

Dog urinary bladder is a target organ of carcinogenic arylamines. However, dog hepatic and urothelial cytosols lack acetylation enzymes that are capable of activating N-hydroxy metabolites of arylamines, suggesting that other enzymes may be involved. In the present study, we found that dog liver microsomes were capable of N-acetylation of 2-aminofluorene and N,O-acetyltransfer of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), and that these activities were inhibited by paraoxon. The 0.25% Triton X-100 extractable fraction of microsomes was resolved on an ion-exchange column into three different proteins that retained these activities. Two of these proteins, designated as enzyme I and enzyme II, were further chromatographed on a Sephacryl S-300 column. As judged from the gel filtration profile, the mol. wt of enzyme I was approximately 180 kDa and that of enzyme II was greater than 700 kDa. SDS-PAGE analysis showed that the subunit weight of enzyme II was approximately 150 kDa. In addition to N-acetylation of 2-aminofluorene and N,O-acetyltransfer of N-OH-AAF, these three enzymes were capable of the deacetylation of 2-acetylaminofluorene, N-OH-AAF and 4-nitrophenyl acetate. The ability of these microsomal enzymes to activate N-hydroxylated aromatic amines and the presence of these enzymes in urothelial cells, reported previously, suggests that they may play an etiological role in the carcinogenicity of these agents in the dog.

Topics: 2-Acetylaminofluorene; Acetylation; Acetyltransferases; Acyltransferases; Amidohydrolases; Animals; Arylamine N-Acetyltransferase; Carcinogens; Chromatography, Ion Exchange; Dogs; Fluorenes; Microsomes, Liver; Nitrophenols; Octoxynol; Polyethylene Glycols

The use of membrane supports as stationary phase, coupled with ligands of choice, allows all kinds of chromatography [Dj. Josić, K. Zeilinger, Y. Lim, M. Raps, W. Hofmann and W. Reutter, J. Chromatogr., 484 (1989) 327] and offers a powerful alternative to both soft gel chromatography and high-performance liquid chromatography. In this work we present affinity membrane chromatography for purification of the enzyme carbonic anhydrase from haemolysates of human erythrocytes. Furthermore, the coupling of the enzymes to the membrane support allows kinetic investigations. As an example, kinetic experiments were carried out by means of carbonic anhydrase coupled to the membrane support using 4-nitrophenyl acetate and 2-chloro-4-nitrophenyl acetate as substrates.

Topics: Carbonic Anhydrases; Chromatography, Affinity; Enzymes, Immobilized; Erythrocyte Membrane; Humans; Kinetics; Membranes, Artificial; Nitrophenols; Solubility; Substrate Specificity

Reacting gastric and pancreatic lipases with mixed diethyl p-nitrophenyl phosphate/bile salt micelles resulted in a stoichiometric inactivation of these enzymes as tested on emulsified tributyroylglycerol and trioleoylglycerol as substrates. Diethyl p-nitrophenyl phosphate treated gastric lipases were also inactive on water-soluble p-nitrophenyl acetate, whereas the modified pancreatic lipase was still able to hydrolyze this water-soluble substrate. The binding of diethyl p-nitrophenyl phosphate modified pancreatic and gastric lipases to tributyroylglycerol/water interface was comparable to that of native lipases. The essential free sulfhydryl group of gastric lipases underwent no chemical changes due to the reaction with micellar diethyl p-nitrophenyl phosphate. All in all, these results indicate that, in both gastric and pancreatic lipases, the essential serine residue which was stoichiometrically labeled by this organophosphorus reagent is involved in catalysis and not in lipid binding.

Topics: Amino Acid Sequence; Animals; Carbon Radioisotopes; Enzyme Activation; Humans; Kinetics; Lipase; Micelles; Molecular Sequence Data; Nitrophenols; Pancreatic Extracts; Pancrelipase; Paraoxon; Rabbits; Substrate Specificity; Triolein

Influenza C virus (strain C/Johannesburg/1/66) was grown, harvested, purified and used as source for the enzyme O-acetylesterase (N-acyl-O-acetylneuraminate O-acetylhydrolase; EC 3.1.1.53). This activity was studied and characterized with regard to some new substrates. The pH optimum of the enzyme is around 7.6, its stability at different pH values shows a result similar to that of the pH optimum, and its activity is well maintained in the pH range from 7.0 to 8.5 (all these tests were performed with 4-nitrophenyl acetate as substrate). Remarkable differences were found in the values of both Km and Vmax, with the synthetic substrates 4-nitrophenyl acetate, 2-nitrophenyl acetate, 4-methylumbelliferyl acetate, 1-naphthyl acetate and fluorescein diacetate. The use of 4-nitrophenyl acetate, 4-methylumbelliferyl acetate or 1-naphthyl acetate as substrate seems to be convenient for routine work, but it is better to carry out the measurements in parallel with those on bovine submandibular gland mucin (the latter is a natural and commercially available substrate). It was found that 4-acetoxybenzoic acid, as well as the methyl ester of 2-acetoxybenzoic acid, but not 2-acetoxybenzoic acid itself, are cleaved by this enzyme. Triacetin, di-O-acetyladenosine, tri-O-acetyladenosine, and di-O-acetyl-N-acetyladenosine phosphate, hitherto unreported as substrates for this viral esterase, are hydrolysed at different rates by this enzyme. We conclude that the O-acetylesterase from influenza C virus has a broad specificity towards both synthetic and natural non-sialic acid-containing substrates. Zn2+, Mn2+ and Pb2+ (as their chloride salts), N-acetylneuraminic acid, 4-methyl-umbelliferone and 2-acetoxybenzoic acid (acetylsalicylic acid) did not act as inhibitors.

Topics: Acetylesterase; Aspirin; Enzyme Stability; Gammainfluenzavirus; Hydrogen-Ion Concentration; Isoflurophate; Kinetics; Lead; Manganese; N-Acetylneuraminic Acid; Nitrophenols; Sialic Acids; Substrate Specificity; Zinc

The colours of frozen solutions containing pH indicators are shown to provide a test for changes in pH in the solvent environment which occur on freezing. Yeast alcohol dehydrogenase loses activity on freezing in phosphate buffer (a buffer in which pH indicator colour changes shows a marked decrease in pH on freezing) but when frozen in bis-tris, Hepes, or N-glycylglycine buffers (all of which show little change in the colour of universal pH indicator and hence of pH on freezing) is stable on freezing. The effects of freezing in different buffer systems on the rate of decomposition of NADPH, and on the rate hydrolysis of 4-nitrophenyl acetate, are rationalised in terms of the pH shifts in these buffers which were determined using universal pH indicator. It is proposed that a major reason for the instability of samples on freezing is the pH changes which occur when some systems are frozen. From the results a general scheme for selecting the best environment for safely freezing samples is proposed which is based on the use of pH indicators.

Topics: Alcohol Dehydrogenase; Colorimetry; Freezing; Hydrogen-Ion Concentration; Indicators and Reagents; NADP; Nitrophenols; Preservation, Biological; Solutions; Water; Yeasts

This paper examines covalent reactivity of AchE with respect to cationic and uncharged methylphosphonates and substrates in the absence and presence of cationic ligands selective for the active center and the peripheral anionic site. The organophosphorus inhibitors are enantiomeric alkyl methylphosphonothioates (1-5) containing cycloheptyl and isopropyl phosphono ester groups and S-methyl, S-n-pentyl, and S-[beta-(trimethylammonio)ethyl] leaving groups; these agents differ in their configuration about phosphorus and their steric, hydrophobic, and electrostatic characteristics. The synthetic substrates examined are acetylthiocholine, p-nitrophenyl acetate, and 7-acetoxy-4-methylcoumarin (7AMC). Antagonism of the methylphosphonothioate reaction by cationic ligands is strongly dependent on the nature of both the cation and the methylphosphonate but independent of the configuration about phosphorus. While all cations cause linear mixed inhibition of acetylthiocholine hydrolysis, there are observed a variety of inhibition patterns of 7AMC and p-nitrophenyl acetate hydrolysis that are distinctly nonlinear, as well as patterns in which the reciprocal plots intersect in the upper right quadrant. Strong antagonism of cationic (methylphosphonyl)thiocholines correlates very well with linear inhibition of acetylthiocholine. Ligands that cause only negligible antagonism of the uncharged methylphosphonates display nonlinear inhibition of uncharged substrates. These relationships, since they are most pronounced for peripheral site ligands and are strongly dependent on the charge carried by the reactant, suggest that the peripheral anionic site alters enzyme reactivity through an electrostatic interaction with the net negative active center. Such behavior indicates a potential role for the peripheral anionic site in conserving AchE catalytic efficiency within a narrow range of values.

Topics: Acetylcholinesterase; Acetylthiocholine; Animals; Binding Sites; Cations; Cholinesterase Inhibitors; Kinetics; Ligands; Models, Chemical; Nitrophenols; Organophosphorus Compounds; Structure-Activity Relationship; Umbelliferones

The present investigation reports the effect of chronic oral administration of mancozeb, a fungicide, on hepatic microsomal carboxylesterases/amidases or B-esterases responsible for hydrolytic metabolism of aspirin (acetylsalicylic acid or ASA) at pH 5.5 and 7.4, 2-acetylaminofluorene (AAF), acetanilide and p-nitrophenylacetate (NPA) and cholinesterase in rat. Oral administration of mancozeb (250 mg/kg/day) for 30 days caused significant stimulation of ASA esterase I (pH 5.5), ASA esterase II (pH 7.4), AAF N-deacetylase and acetanilide N-deacetylase in liver. However, the activities of NPA esterase and cholinesterase remained unaffected. Evaluation of induction kinetics demonstrated that the pattern and magnitude of responses of these microsomal hydrolases to mancozeb treatment for 7 days were comparable to those obtained after treatment for 30 days. The activities of hydrolases were not altered in animals killed 4 hr after an oral dose of mancozeb. Mancozeb did not affect these hydrolases in vitro.

Topics: 2-Acetylaminofluorene; Acetanilides; Animals; Aspirin; Fungicides, Industrial; Hydrolases; Male; Maneb; Microsomes, Liver; Nitrophenols; Rats; Xenobiotics; Zineb

1. The kinetics and inhibition of p-nitrophenylacetate hydrolysis by cytosolic esterases of female alfalfa leafcutting bees, Megachile rotundata (Fab.) was examined. 2. For p-nitrophenylacetate, the Km = 1.24 x 10(-4) M and Vmax = 2.29 x 10(-9) mol/s per mg protein. 3. Regarding four organophosphate insecticides, the mechanism of inhibition in all cases was mixed (competitive and uncompetitive) and, based on inhibition constants, the order of toxicity was naled greater than paraoxon greater than trichlorfon greater than oxydemeton methyl. 4. Comparisons are made to the honey bee, Apis mellifera.

Topics: Animals; Bees; Esterases; Female; Hydrolysis; Insecticides; Kinetics; Naled; Nitrophenols; Organothiophosphorus Compounds; Paraoxon; Trichlorfon

Hydrolytic reactions in the presence of liposomes catalyzed by N epsilon-benzyloxycarbonylhistidine groups introduced into the side chains of poly[N-(3-aminopropyl)glycine] were studied. On increasing the hydrophobicity of the polypeptide catalyst by introducing dodecyl groups into the side chains, and in the presence of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes, p-nitrophenyl palmitate (PNPP) was hydrolyzed more rapidly than p-nitrophenyl acetate (PNPA). The addition of cholesterol or phosphatidylserine to lipid bilayer membranes accelerated the hydrolysis of PNPP catalyzed by the polypeptide catalyst more strongly than that of PNPA. The substrate selectivity and catalytic efficiency of the polypeptide catalyst were found to be controlled by the physical state of the lipid bilayer membranes.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Catalysis; Chemical Phenomena; Chemistry, Physical; Cholesterol; Hydrolysis; Imidazoles; Lipid Bilayers; Liposomes; Nitrophenols; Palmitates; Palmitic Acids; Peptides; Phosphatidylserines; Polymers; Propylamines

A variety of chemically defined compounds were tested to characterize the substrate specificity of the influenza C virus esterase and to determine whether a substrate could be found that would be useful in an assay to detect the virus. Two new substrates, alpha-naphthyl acetate and alpha-naphthyl propionate, were identified; alpha-naphthyl acetate was employed to develop an assay specific for influenza type C virus in MDCK cells. The assay was sufficiently sensitive to detect esterase activity in a single cell and distinguished influenza C virus infections from those of types A and B viruses. Infected cells could be detected as early as 8 h postinfection, with maximal enzyme detection occurring at 24 h. Assay of influenza C virus in the chorioallantoic or amniotic fluid of infected eggs was performed by applying fluids directly onto nitrocellulose strips and then incubating with alpha-naphthyl acetate. Both the cellular and nitrocellulose-bound assays are rapid, inexpensive, and easy to perform, offering advantages for use in clinical laboratories.

Topics: Animals; Chick Embryo; Esterases; Gammainfluenzavirus; Hydrogen-Ion Concentration; Hydrolysis; Naphthalenes; Naphthols; Nitrophenols; Orthomyxoviridae; Propionates; Substrate Specificity; Temperature

The bile-salt-dependent lipase from human milk, which catalyzes the hydrolysis of the water-soluble substrate 4-nitrophenyl acetate and the water-insoluble substrate tributyrin, is competitively inhibited by phenyl boronic acid. This inhibitor does not interfere with the interaction of lipase either with the siliconized glass beads/water interface or with the activator bile-salt binding site. The boronic acid binds near or at the active site serine, since modification of this residue by diisopropylphosphofluoridate (DFP) was prevented by phenyl boronic acid. Phenyl boronic acid binds 15-fold as tightly to bile-salt-dependent lipase as does 4-nitrophenyl acetate. Therefore, phenyl boronic acid bears analogy to a substrate rather than to a tetrahedral intermediate analog. Bile salts such as sodium taurocholate which are non-essential activators for the milk lipase activity on water-soluble substrates decrease the Km as well as the enzyme inhibitor dissociation constant (Ki). They have a slight effect on kcat. These results are interpreted in terms of an increase of the stability of the enzyme-substrate tetrahedral intermediate and in general of any transition states for the formation and for the decomposition of these intermediates upon the enzyme bile salts interaction.

Topics: Adsorption; Binding Sites; Boronic Acids; Glass; Humans; Hydrogen-Ion Concentration; Isoflurophate; Kinetics; Lipase; Milk, Human; Nitrophenols; Silicon; Sterol Esterase; Taurocholic Acid; Triglycerides

Kinetic and thermodynamic parameters were evaluated for the acylation and the deacylation steps in the hydrolysis of p-nitrophenyl acetate by alpha-chymotrypsin at pH 7.8 and at temperatures between 15 and 35 degrees C by the use of stopped-flow and ordinary ultraviolet spectrophotometers. In contrast to the temperature dependencies of k2 and Ks reported in the literature (P.A. Adams and E.R. Swart, Biochem. J., 161, 83 (1977], no kinetic anomaly was observed in either of the steps, but reasonable straight lines were obtained in both Arrhenius and van't Hoff plots. On the other hand, in the chymotryptic hydrolysis of N-benzoyl-L-alanine methyl ester a sharp kinetic anomaly was found. The discrepancy in the case of p-nitrophenyl acetate is discussed in connection with a possible conformational change of the enzyme, an alteration of the rate-limiting step or differences in the experimental procedures. The cause of the anomaly observed in the case of N-benzoyl-L-alanine methyl ester is also discussed in detail.

Topics: Alanine; Chymotrypsin; Hydrolysis; Nitrophenols; Thermodynamics

The reactions of lipase (449 amino acid residues) and lipase fragment (336-449) with p-nitrophenyl acetate have been studied from 2 different angles. In previous papers it has been shown that lipase and lipase fragment enzymatically hydrolyze p-nitrophenyl acetate. The amino acid residue of the catalytic site that is temporarily acetylated has not yet been characterized in lipase or lipase fragment. Besides this very fast enzymatic hydrolysis, acetylation reactions may take place on nucleophilic amino acid side-chain groups. In the present report, acetylated amino acid residues whose acetyl linkages were not cleaved after pH 7.5-8.5 incubations have been investigated. Several residues were acetylated in very low proportion, whereas lysine 373 was stoichiometrically acetylated in lipase and in lipase fragment. This specific acetylation may have been favored by the presence of a hydrophobic reversible binding site for p-nitrophenyl acetate near Lys-373. This acetylation did not greatly change the specific activity of lipase towards an emulsion of tributyrylglycerol in the presence of colipase, but under certain conditions it had an effect on the enzymatic hydrolysis of p-nitrophenyl acetate by the lipase fragment.

Topics: Acetylation; Amino Acid Sequence; Animals; Chromatography, Gel; Enzyme Stability; Hydrogen-Ion Concentration; Hydrolysis; Lipase; Lysine; Nitrophenols; Pancreas; Rabbits; Swine

The structures of sugar chains of a p-nitrophenyl acetate-hydrolyzing esterase from the microsomes of rat liver were established. The enzyme contained mannose and glucosamine as sugar components. Asparagine-linked sugar chains of the esterase were liberated by hydrazinolysis. After N-acetylation of the hydrazinolysate, the reducing ends of the sugar chains were coupled with 2-aminopyridine. Fluorescent pyridylamino (PA-) derivatives of sugar chains thus obtained were purified by gel filtration and reversed-phase HPLC. Eleven PA-sugar chains were obtained. The structures of the PA-sugar chains were first identified by HPLC using two series of separation systems by which 11 PA-oligomannose-type sugar chains with known structures could be separated. Further elucidation of the structures of each PA-sugar chain was performed by exoglycosidase digestions and partial acetolysis. The structures of two of the PA-sugar chains were further confirmed by 500 mHz 1H-NMR spectroscopy.

Topics: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Carbohydrates; Chromatography, Gel; Chromatography, High Pressure Liquid; Esterases; Glycoside Hydrolases; Hydrazines; Magnetic Resonance Spectroscopy; Microsomes, Liver; Molecular Sequence Data; Nitrophenols; Protein Conformation; Rats

Acylation of the aldehyde dehydrogenase.NADH complex by acetic anhydride leads to the production of acetaldehyde and NAD+. By monitoring changes in nucleotide fluorescence, the rate constant for acylation of the active site of the *enzyme.NADH complex was found to be 11 +/- 3 s-1. The rate of acylation by acetic anhydride at the group that binds aldehydes on the oxidative pathway is clearly rapid enough to maintain significant steady-state concentrations of the required active-site-acylated *enzyme.NADH intermediate despite the rapid hydrolysis of this *enzyme.acyl.NADH intermediate (5-10 s-1) [Blackwell, Motion, MacGibbon, Hardman & Buckley (1987) Biochem. J. 242, 803-808]. Hence reversal of the normal oxidative pathway can occur. However, although acylation of the aldehyde dehydrogenase.NADH complex by 4-nitrophenyl acetate also occurs rapidly with a rate constant of 10.9 +/- 0.6 s-1, even under the most extreme trapping conditions only very small amounts of acetaldehyde are detected [Loomes & Kitson (1986) Biochem. J. 235, 617-619]. Furthermore enzyme-catalysed hydrolysis of 4-nitrophenyl acetate is limited by the rate of deacylation of a group on the enzyme (0.4 s-1), which is an order of magnitude less than deacylation of the group at the active site (5-10 s-1). It is concluded that the enzyme-catalysed 4-nitrophenyl ester hydrolysis involves a group on the enzyme that is different from the active-site group that binds aldehydes on the normal oxidative pathway.

Topics: Acetaldehyde; Acetic Anhydrides; Acylation; Aldehyde Dehydrogenase; Aldehydes; Binding Sites; Hydrolysis; Macromolecular Substances; NAD; Nitrophenols; Oxidation-Reduction

1. Serum albumins from nine of 10 vertebrate species were found to react rapidly with p-nitrophenylacetate. 2. The high reactivities were shown to be partially attributable to strong, rapidly reversible binding of p-nitrophenylacetate by each serum albumin. 3. As previously observed in the case of human serum albumin (Koh and Means, Arch. Biochem. Biophys. 192, 73-79, 1979), this binding takes place in the primary binding site for several physiologically (i.e. tryptophan, small fatty acid anions) and pharmacologically (i.e. diazepam) important compounds. 4. Horse serum albumin differed from all other serum albumins included in this study in that it did not react rapidly with p-nitrophenylacetate, presumably, due to significant differences in its corresponding binding site.

Topics: Amino Acid Sequence; Animals; Humans; In Vitro Techniques; Molecular Sequence Data; Nitrophenols; Protein Binding; Serum Albumin; Species Specificity; Vertebrates

Aldehyde dehydrogenase can catalyze the hydrolysis of esters such as p-nitrophenyl acetate as well as oxidize aldehydes to acids. It has not been proven unequivocally that the two reactions occur at the same active site. In the accompanying paper (Tu, G. C., and Weiner, H. (1988) J. Biol. Chem. 263, 1212-1217) evidence was presented which showed that cysteine at position 49 was at the active site for the dehydrogenase reaction. Evidence also was presented which showed that cysteine located at position 162 was susceptible to modification by N-ethylmaleimide. It was shown here that the two activities of the enzyme can be differently protected from inactivation by substrate analogs. Furthermore, aldehydes were found to be poor inhibitors against the esterase reaction while ester was a good inhibitor against the dehydrogenase reaction. In addition, it was possible to modify cysteine 49 with N-ethylmaleimide but not find inhibition of the esterase reactivity until cysteine 162 was modified. It appears that horse liver aldehyde dehydrogenase has two separate active sites per subunit. The data fit a model where ester can be hydrolyzed at both sites but that aldehyde oxidation occurred only at position 49.

Topics: Aldehyde Dehydrogenase; Animals; Binding Sites; Chromatography, High Pressure Liquid; Ethylmaleimide; Horses; Kinetics; Magnesium; Mitochondria, Liver; Nitrophenols; Phenylmethylsulfonyl Fluoride

Macrophages from both rodent and human sources have been shown to produce lipoprotein lipase (LPL), the enzyme activity of which can be measured in culture media and in cellular homogenates. The studies reported here show the presence of LPL on the surface of human monocyte-derived macrophages. An inhibitory monoclonal antibody to human LPL was used for cellular and immunoelectron microscopy studies. This antibody is a competitive inhibitor of LPL hydrolysis of triacylglycerol but does not inhibit LPL hydrolysis of a water-soluble substrate, p-nitrophenyl acetate. Furthermore, when postheparin plasma was mixed with monoclonal antibody prior to gel filtration on 6% agarose, the LPL activity eluted with the lipoproteins and was not inhibited by the antibody. These studies suggest that the antibody recognized the lipid/lipoprotein binding site of the LPL molecule. Membrane-bound LPL was demonstrated on human monocyte-derived macrophages using colloidal gold-protein A to detect the monoclonal antibody to LPL. The surface colloidal gold was randomly distributed with a surface density of 56,700 gold particles per cell. Control cells cultured in heparin-containing media (10 units/ml) or cells reacted with anti-hepatic triacylglycerol lipase monoclonal IgG or nonimmune mouse IgG did not exhibit membrane binding of protein A-gold. Macrophages were incubated with control and monoclonal anti-LPL IgGs and 125I-labeled anti-mouse IgG F(ab')2. Heparin-releasable membrane-bound anti-LPL antibody was demonstrated. These studies demonstrate the presence of LPL on the surface of human monocyte-derived macrophages, such that the LPL is oriented with its lipid-binding portion (recognized by this antibody) exposed. Membrane-associated LPL may be important in the interaction and subsequent uptake of lipid and lipoproteins by macrophages and in the generation of atherosclerotic foam cells.

Topics: Antibodies, Monoclonal; Cell Adhesion; Humans; Immunohistochemistry; Lipoprotein Lipase; Macrophages; Nitrophenols

With and without p-chlorophenol as an activator, the rates of hydrolysis of p-nitrophenyl acetate catalyzed by alpha-chymotrypsin were measured at pressures up to 2 kbar at 25 degrees C. From the pressure dependence of the rate constant (kcat)A and (kcat)0 of the product formation with and without an activator, the activation volumes (delta V not equal to cat)A and (delta not equal to cat)0 were +2 and -6 +/- 1 cm3.mol-1. From the pressure dependence of the equilibrium constant (KA) of incorporation of p-chlorophenol into the enzyme, the volume change (delta VA) was -10 +/- 1 cm3.mol-1. The mechanisms of the substrate activation are discussed in terms of the activation and reaction volumes.

Topics: Atmospheric Pressure; Catalysis; Chlorophenols; Chymotrypsin; Enzyme Activation; Kinetics; Nitrophenols; Protein Conformation

A ribosylated derivative of adenine, N6 ribosyl adenine, likely to have formed under prebiotic synthesis conditions, is shown to be as active as histidine in the model reaction of p-nitrophenyl acetate hydrolysis. This property widens the range of reactions accessible to RNA catalysis.

Topics: Adenosine; Catalysis; Histidine; Hydrolysis; Nitrophenols; RNA

A series of substituted trifluoroketones were tested as inhibitors of mammalian liver microsomal carboxylesterase(s) hydrolyzing a variety of substrates including malathion, diethylsuccinate (DES) and p-nitrophenyl acetate (p-NpAc). The trifluoroketones used were very potent "transition state" inhibitors of crude mouse and human liver microsomal carboxylesterases as well as commercial porcine liver carboxylesterase (Sigma EC 3.1.1.1 Type I). These enzymes were found to differ in their sensitivity to the inhibitors employed, and some compounds caused dramatic activation of the hydrolysis of DES. In some but not all cases, a thioether beta to the carbonyl increased the inhibitory potency of the compound. Structure-activity relationships also were evaluated among aliphatic versus substituted and unsubstituted aromatic trifluoroketones. Kinetic parameters [i.e. Km, Vmax and (T1/2)e] for the mouse liver microsomes and the porcine carboxylesterase hydrolyzing DES were determined. Apparent high- and low-affinity forms were observed with each preparation. 3-Nonylthio-1,1,1-trifluoropropan-2-one was synthesized by the reaction of the corresponding thiol with 3-bromo-1,1,1-trifluoroacetone, and apparent synergism was observed when it was coadministered i.p. with malathion to mice.

Topics: Animals; Carboxylic Ester Hydrolases; Ketones; Kinetics; Malathion; Mice; Microsomes, Liver; Nitrophenols; Species Specificity; Structure-Activity Relationship; Succinates; Swine

Topics: Binding Sites; Esterases; Humans; Nitrophenols; Pharmaceutical Preparations; Protein Binding; Serum Albumin

Treatment with 0.5% (w/w) dietary clofibrate, a peroxisome proliferator, for 14 days induced microsomal carboxylesterase activities for five substrates including malathion, clofibrate, diethylsuccinate, diethylphthalate, and p-nitrophenylacetate in liver and kidney of male Swiss-Webster mice and Sprague-Dawley rats. The induction was substrate, tissue, and species dependent. The carboxylesterase activity was induced in mouse from 1.2- to 2.2-fold (liver) and from 1.1- to 1.7-fold (kidney) depending upon substrate used. Analogous values from rat ranged from 1.0- to 1.4-fold (liver) and from 1.1- to 1.8-fold (kidney). Enzyme activities were either decreased or not affected in testes of treated mice and rats. Substituted trifluoroketones ("transition-state" inhibitors of carboxylesterase) were found to be very potent inhibitors of clofibrate-metabolizing carboxylesterase(s) and to be potentially useful in distinguishing among isozymes. The inhibition data suggested that changes in carboxylesterase activity following clofibrate treatment were both qualitative and quantitative.

Topics: Animals; Carboxylic Ester Hydrolases; Clofibrate; Enzyme Induction; Kidney; Malathion; Male; Mice; Microsomes; Microsomes, Liver; Nitrophenols; Phthalic Acids; Rats; Rats, Inbred Strains; Succinates; Testis

(1) The interaction of bile-salt-stimulated human milk lipase and liposomal membranes has been investigated in the presence or absence of sodium taurocholate. Freshly purified enzyme enhances the permeability of liposomal membranes but thermally inactivated enzyme does not. (2) The ability of the enzyme to catalyze the hydrolysis of a relatively hydrophilic substrate, 4-nitrophenyl acetate, and a more hydrophobic substrate, 4-nitrophenyl palmitate, has also been measured in media containing small unilamellar vesicles of egg phosphatidylcholine in both the absence and presence of taurocholate, and also in the presence of free taurocholate in the absence of liposomes. (3) The enzyme-catalyzed hydrolysis of 4-nitrophenyl acetate is enhanced in all of these systems, but 4-nitrophenyl palmitate is protected from enzymic attack in the phosphatidylcholine-bile salt systems. If free taurocholate be present in the system before 4-nitrophenyl palmitate is added, then, and only then, is enzymic activity observed. (4) These results have been interpreted in terms of the importance of the microenvironment around the substrate and the role played by the bile salt surfactant in stimulating the enzyme.

Topics: Bile Acids and Salts; Colloids; Enzyme Activation; Female; Humans; Lipase; Liposomes; Micelles; Milk, Human; Nitrophenols; Palmitates; Phosphatidylcholines; Taurocholic Acid

We report three experiments which show that the hydrolysis of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from bovine skeletal muscle occurs at a site on the enzyme different than the active site for CO2 hydration. This is in contrast with isozymes I and II of carbonic anhydrase for which the sites of 4-nitrophenyl acetate hydrolysis and CO2 hydration are the same. The pH profile of kcat/Km for hydrolysis of 4-nitrophenyl acetate was roughly described by the ionization of a group with pKa 6.5, whereas kcat/Km for CO2 hydration catalyzed by isozyme III was independent of pH in the range of pH 6.0-8.5. The apoenzyme of carbonic anhydrase III, which is inactive in the catalytic hydration of CO2, was found to be as active in the hydrolysis of 4-nitrophenyl acetate as native isozyme III. Concentrations of N-3 and OCN- and the sulfonamides methazolamide and chlorzolamide which inhibited CO2 hydration did not affect catalytic hydrolysis of 4-nitrophenyl acetate by carbonic anhydrase III.

Topics: Animals; Apoenzymes; Carbon Dioxide; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Cattle; Hydrogen-Ion Concentration; Hydrolysis; Isoenzymes; Kinetics; Muscles; Nitrophenols

The presence of non-specific esterase activity is correlated with different Leydig cell characteristics: 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), human chorionic gonadotrophin binding and LH-stimulated steroid production. This indicates that esterase can be used as a marker enzyme for Leydig cells. Esterase, however, has also been used as a marker enzyme for macrophages. We have compared, using biochemical and histochemical techniques, the esterase activity of Leydig cell preparations from mature and immature rats and of preparations enriched in testicular or peritoneal macrophages. Leydig cells were identified by staining for 3 beta-HSD, and macrophages by phagocytosis of fluorescent beads. Leydig cell preparations from mature rats showed an approximately 400-fold higher esterase activity than peritoneal macrophage preparations and an approximately 50-fold higher activity than testicular macrophage preparations. Leydig cell preparations from mature rats showed a 60-fold higher esterase activity than Leydig cell preparations from immature rats. Differences in esterase activity were also demonstrated histochemically. Leydig cells from mature rats showed positive esterase staining after 30 s at room temperature. Testicular macrophages showed esterase activity after staining for 3 min. Only approximately 25% of the 3 beta-HSD-positive cells from immature rats showed esterase activity after staining for 6 min. Esterase is therefore a useful marker enzyme for Leydig cells from mature rats and can be of help in studies concerning the development of these cells.

Topics: 3-Hydroxysteroid Dehydrogenases; Animals; Carboxylesterase; Carboxylic Ester Hydrolases; Histocytochemistry; Leydig Cells; Macrophages; Male; Nitrophenols; Peritoneal Cavity; Phagocytosis; Rats; Rats, Inbred Strains; Testis

Subunit III has so far been found only in the pancreas of ruminants in a non-covalent association (procarboxypeptidase A-S6) with two different proteins: the procarboxypeptidase A itself (subunit I) and a C-type chymotrypsinogen (subunit II). In contrast with these latter two proteins, which are zymogens of pancreatic proteases, subunit III seems to be devoid of any activity towards specific substrates of pancreatic proteases. However, it possesses a weakly functional active site which allows it to hydrolyze a non-specific ester, p-nitrophenyl acetate, and to react with several active-site titrants. The binding of proflavin to subunit III shows that this protein owns a non-polar binding site with a very high Kd compared to that of chymotrypsin. The comparison of the amino acid sequences of subunit III and some serine proteases showed that subunit III is closely related to an elastase. Models of the tertiary structure of subunit III suggest a conformational modification that affects the substrate binding and could explain the lack of specific enzymatic activity. The presence of subunit III in the ternary complex is not related to an enzymatic function. This protein does not participate in the activation process of subunit I but prevents the denaturation of this subunit at low pH. This may represent its biological role in the acidic environment of the duodenum in ruminants.

Topics: Amino Acid Sequence; Animals; Binding Sites; Carboxypeptidases; Carboxypeptidases A; Cattle; Enzyme Precursors; Esterases; Goats; Hydrogen-Ion Concentration; Nitrophenols; Pancreas; Pancreatic Elastase; Proflavine; Protein Conformation; Sheep; Trypsin

The incubation of porcine pancreatic lipase (449 amino acids) with chymotrypsin led to the preferential cleavage of the Phe-335-Ala-336 bond [Bousset-Risso et al. (1985) FEBS Lett. 182, 323-326]. Up to now it has not been possible to isolate the fragment (1-335) whereas fragment (336-449) was purified. This fragment does not display any activity towards the specific substrates of lipase, triacylglycerols, either in the aggregate form or monomeric solution, but like lipase it hydrolyzes p-nitrophenyl acetate. The biphasic kinetics of the release of p-nitrophenol by the fragment with different concentrations of p-nitrophenyl acetate ([S] greater than [E]) are very similar to those of lipase and other esterases. The initial burst is equal to 1 mol p-nitrophenol/mol fragment (when [S] = infinity). Ethoxyformic anhydride only reacts with 1 mol histidine out of the 2 mol that the fragment contained. The activity of the fragment towards p-nitrophenyl acetate hydrolysis is inhibited after ethoxyformic anhydride reaction as in the case of lipase. The results presented led to the hypothesis that in the area (336-449) a part of the active-site structure of the lipase molecule is included. It would seem that fragment (336-449) is a functional domain of lipase.

Topics: Acetylation; Animals; Binding Sites; Diethyl Pyrocarbonate; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Lipase; Nitrophenols; Pancreas; Peptide Fragments; Swine

Topics: Enzymes; Hydrolysis; Models, Biological; Nitrophenols; Oligopeptides; Peptides; Structure-Activity Relationship

Incubation of sheep liver cytoplasmic aldehyde dehydrogenase with the substrate 4-nitrophenyl [14C]acetate in the presence of NADH leads to the formation of 14C-labelled acetaldehyde. This observation strongly supports the idea that the esterase and dehydrogenase activities of the enzyme occur at the same site and involve the intermediacy of a common acyl-enzyme.

Topics: Acetaldehyde; Aldehyde Dehydrogenase; Animals; Chromatography, Thin Layer; NAD; Nitrophenols; Semicarbazides; Sheep

The protein ubiquitin undergoes extensive N epsilon-acetylation of some of its seven lysine residues when reacted with p-nitrophenyl acetate. Lysines 27 and 29 show little reactivity whereas residue 6 is the most readily acetylated. Residues 11, 33, 48, and 63 show intermediate reactivities.

Topics: Acetylation; Amino Acids; Animals; Buffers; Cattle; Chemical Phenomena; Chemistry; Electrophoresis, Disc; High Mobility Group Proteins; Horses; Lysine; Malonates; Nitrophenols; Peptide Fragments; Ubiquitins

Cyanogen (C2N2), a molecule with properties remarkably similar to carbon dioxide, differentially inhibits three of the four carbonic anhydrases reported here. Bovine carbonic anhydrase II shows 97% loss of esterase activity with no concommitant loss in hydratase activity. The hydratase and esterase activities of human carbonic anhydrase I are decreased by 80% and 55% respectively. Canine carbonic anhydrase shows similar results to human carbonic anhydrase I, retaining 29% hydratase and 62% esterase activity. Rabbit carbonic anhydrase sustained no loss of either hydratase or esterase activity. This inhibition occurs by an irreversible modification of the enzymes. The kinetic parameters for modified and unmodified enzymes were altered in a way that reflects the characteristic effect for each carbonic anhydrase.

Topics: Acetates; Acetazolamide; Animals; Carbon Dioxide; Carbonic Anhydrase Inhibitors; Cattle; Dogs; Humans; Iodoacetamide; Iodoacetates; Iodoacetic Acid; Kinetics; Nitriles; Nitrophenols; Pyruvates; Rabbits

Ten murine monoclonal antibodies have been produced that are specific for bovine milk lipoprotein lipase. One monoclonal antibody, bLPL-mAb-7, inhibited completely the apolipoprotein C-II (apo-C-II)-dependent enzymic hydrolysis of trioleoylglycerol in a phospholipid-stabilized emulsion, but had no effect on the hydrolysis of the water-soluble substrate p-nitro-phenylacetate. Four times more bLPL-mAb-7 was required to achieve 50% inactivation of lipoprotein lipase activity when the enzyme was preincubated with excess apo-C-II. Disruption of the binding of a dansyl-labeled apo-C-II peptide to lipoprotein lipase by bLPL-mAb-7 was demonstrated by resonance energy transfer, both in the presence and absence of lipid. This antibody thus appears to recognize the apo-C-II binding site of lipoprotein lipase. In addition, bLPL-mAb-7 also inhibited the lipoprotein lipase activity of human post-heparin plasma.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Apolipoprotein C-II; Apolipoproteins C; Binding Sites; Energy Transfer; Heparin; Hydrolysis; Lipoprotein Lipase; Male; Mice; Mice, Inbred BALB C; Milk; Nitrophenols; Triglycerides

The present study has provided evidence for the existence of three distinct carboxylesterases involved in the hydrolysis of steroid esters, where two enzymes are possibly responsible for the metabolism of hydrocortisone hemisuccinate (HCHS) at pH 5.5 and 8.0, and a third enzyme for the metabolism of hydrocortisone acetate (HCAC) at pH 8.0, in isolated rat liver microsomes. The activity of all three enzymes in rat liver was induced significantly by the administration of phenobarbital while no such function in enzyme activity was observed in animals receiving 3-methylcholanthrene or benzo[a] pyrene under similar experimental conditions. The increase in the activity of HCHS esterase I (HCHS-E1) active at pH 5.5, HCHS esterase II (HCHS-E2) active at pH 8.0, and HCAC esterase (HCAC-E) was approximately 7 to 8, 3- and 3-fold respectively. On the other hand, the degree of induction of nonspecific microsomal carboxylesterase acting on p-nitrophenylacetate (PNPA) was significantly less. The Km values for the hydrolysis of HCHS at pH 5.5 and 8.0 and HCAC by rat liver microsomes obtained from control rats were 2.45, 2.02 and 1.6 mM, respectively, and these Km values were not changed significantly in preparations obtained from rats treated with phenobarbital. The distinct in vitro responses displayed by hepatic microsomal steroid esterases to various inhibitors were able to distinguish three different enzymes which also differed from nonspecific carboxylesterases. The activity of HCAC-E was inhibited by NaAsO2 and AgNO3 while that of HCHS-E1 and HCHS-E2 remained unaffected. Selective inhibition of HCHS-E1 by NaF, HgCl2 and p-chloromercuribenzoate and that of HCHS-E2 by NiSO4 indicated the possible existence of different enzymes or isozymes of a carboxylesterase catalyzing HCHS hydrolysis. The effects elicited by the inhibitors on the activity of PNPA esterase were different from those observed with steroid esterases. Furthermore, the present study has also indicated species variations in the distribution of steroid esterases in the livers of rat, mouse, dog and cat.

Topics: Animals; Carboxylic Ester Hydrolases; Female; Hydrocortisone; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Microsomes, Liver; Nitrophenols; Rats; Species Specificity

The binding of oxazepam and its glucuronide conjugates to human serum albumin (HSA), as well as the binding interactions of the drug and its metabolites, were examined by equilibrium dialysis and kinetic probe studies. Oxazepam and its S(+) glucuronide are bound to the HSA molecule with affinity constants of 3.5 X 10(5) M-1 and 5.5 X 10(4) M-1, respectively, which were independent of protein concentration over a range of 0.1 to 5.0 g/dl. The R(-) glucuronide bound weakly to albumin, with the binding parameter, N X K, increasing at lower albumin concentrations. Pre-acetylation of fatty acid free-HSA resulted in decreased binding of all three compounds, probably by altering the conformation of the binding sites. Kinetic probe studies with p-nitrophenyl acetate indicate that oxazepam and its S(+) glucuronide shared a common binding site on HSA, but that the R(-) glucuronide bound at another site. Oxazepam binding was unaffected by the presence of its glucuronide conjugates but was inhibited by fatty acids. The percentage of oxazepam bound to plasma proteins in patients with renal impairment (94%) was lower than in normal volunteers (97%). This lower binding can neither be attributed to lower albumin concentrations because of the large binding capacity of the protein and linearity of N X K nor to displacement by elevated concentrations of glucuronide conjugates, but it may be ascribed partly to increased plasma fatty acids.

Topics: Glucuronates; Humans; In Vitro Techniques; Kidney Diseases; Nitrophenols; Oxazepam; Protein Binding; Serum Albumin; Stereoisomerism

The evidence for and against the esterase and dehydrogenase active sites of aldehyde dehydrogenase being topologically distinct is examined. It is found that all the evidence (including all that previously amassed by others in favour of distinct binding domains) is actually consistent with, and in favour of, a single type of catalytic site having both activities. The existence of separate high-Km modulating sites for the enzyme is also questioned.

Topics: Aldehyde Dehydrogenase; Binding Sites; Hydrolysis; Kinetics; Macromolecular Substances; NAD; Nitrophenols

The possible existence of intramolecular interactions involving the tyrosine and histidine residues in angiotensin II has been investigated by measuring the reactivities of the functional groups in the molecule. Angiotensin II catalyzed the hydrolysis of p-nitrophenylacetate in the pH range 6.6-8.2 at higher rates than were consistent with the reactivities of the free constituent functional groups, and had 2-4% of the activity of chymotrypsin between pH 6.6 and 7.5. Treatment of angiotensin II with acetic anhydride demonstrated that the tyrosine hydroxyl and the imidazole side-chain in angiotensin II acetylated and deacetylated at markedly higher rates than for the free amino acids, indicating increased nucleophilicities and the presence of intrinsic deacetylation mechanisms for these residues in angiotensin II. These findings are consistent with the presence of tyrosine hydroxyl-histidine-carboxylate charge relay system in ANG II in aqueous environments, and suggest that ANG II may act at membrane receptors by a mechanism which is analogous to that operating in serine proteases.

Topics: Acetic Anhydrides; Acetylation; Angiotensin II; Chemical Phenomena; Chemistry; Dealkylation; Histidine; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Nitrophenols; Protein Conformation; Spectrophotometry, Infrared; Tyrosine

The goal of this investigation was to determine the reason for the previously reported increase in the rate of hydrolysis of p-nitrophenyl acetate to p-nitrophenol in the presence of positively charged liposomes. When this charge was due to incorporation of stearylamine, the rate of loss increased 5- to 10-fold relative to the control buffers. This rate enhancement was accompanied by formation of N-stearylacetamide, an event which was not previously considered. Similar results were obtained with either L-alpha- or dimyristoyl phosphatidylcholine. When the positive charge on the liposomes was conferred by the cetrimonium ion, however, the acceleration was replaced by a reduction in rate together with the absence of amide formation. Separation of the continuous phases from the liposomes provided media which were kinetically equivalent to the control buffers, indicating that rate enhancement and reduction were both due to the liposomal phases. Increasing the pH produced an increase in ester clearance values due to the stearylamine-containing liposomal phase, which is consistent with the formation of free amine, providing increased aminolysis. Although amide formation was also observed in stearylamine suspensions, the rate of p-nitrophenyl acetate loss was much greater in liposomal suspensions. Accelerated loss in the presence of positively charged liposomes is due to the formation of N-stearylacetamide by reaction with stearylamine and not to the positive charge, a hypothesis disproved by use of cetrimonium ion containing liposomes.

Topics: Amines; Buffers; Cetrimonium Compounds; Chromatography, Thin Layer; Dimyristoylphosphatidylcholine; Drug Stability; Hydrolysis; Kinetics; Liposomes; Nitrophenols; Particle Size; Phosphatidylcholines; Suspensions

Hepatic and extrahepatic carboxylesterase (EC 3.1.1.1) activities were studied after the exposure of rats to polycyclic aromatic hydrocarbons. In dose- and time-dependent studies, the carcinogens benz[a]anthracene, benzo[a]pyrene and 3-methylcholanthrene moderately induced the hepatic cytosolic and kidney microsomal carboxylesterase activities, while the non-carcinogenic compounds anthracene, phenanthrene and chrysene had no effects on these enzyme activities. The hepatic microsomal and kidney cytosolic enzyme activities were not altered by the polycyclic aromatic hydrocarbons tested. Carboxylesterase activity in the postmitochondrial fraction of the intestinal mucosa decreased the serum enzyme activity slightly increased after the administration of the carcinogenic compounds. Although the in vitro hydrolysis of propanidid by hepatic cytosolic and kidney microsomal preparations was increased, the biological half-life of propanidid in vivo was not changed by 3-methylcholanthrene treatment.

Topics: Animals; Anthracenes; Benz(a)Anthracenes; Benzo(a)pyrene; Benzopyrenes; Carboxylic Ester Hydrolases; Chrysenes; Enzyme Induction; Intestinal Mucosa; Kidney; Liver; Male; Methylcholanthrene; Nitrophenols; Phenanthrenes; Polycyclic Compounds; Rats; Rats, Inbred Strains

Ubiquitin was isolated from bovine erythrocytes by a relatively simple procedure involving extraction with chloroform and ethanol, chromatography on DEAE-cellulose, and gel filtration. Amino acid and partial sequence analyses showed it to be identical to previously isolated material. Ubiquitin released p-nitrophenolate from p-nitrophenyl acetate, but did not cleave other esterase substrates that were tested. It had a turnover number of 116 mmol for p-nitrophenyl acetate at pH 7.7 and 30 degrees C, and this activity was relatively stable to heat treatment. Electrophoretic studies indicated that the ubiquitin was sequentially acetylated by p-nitrophenyl acetate, as judged by the appearance of more anodically migrating components. The reactions of ubiquitin with p-nitrophenyl acetate at pH 7.0 were biphasic and consisted of (a) an initial phase, during which the release of p-nitrophenol resulted from monoacetylation of the ubiquitin and from ubiquitin-catalyzed hydrolysis of the ester; and (b) a second phase, during which the release of p-nitrophenol resulted only from the breakdown and reformation of the acetyl-enzyme complex. Ubiquitin also showed CO2 hydration activity and could be localized following gel electrophoresis by the CO2-bromthymol blue staining method. The strong inhibitor of carbonic anhydrase, acetazolamide, also inhibited the CO2 hydration activity and p-nitrophenyl acetate activity of ubiquitin. An antibody against this protein did not precipitate bovine carbonic anhydrase II. The esterase activity of ubiquitin was much higher than those previously reported for the carbonic anhydrases.

Topics: Acetylation; Animals; Carbon Dioxide; Cattle; Chromatography, DEAE-Cellulose; Chromatography, Gel; Erythrocytes; Esterases; High Mobility Group Proteins; Hot Temperature; Hydrolysis; Immunodiffusion; Nitrophenols; Peptide Fragments; Trypsin; Ubiquitins

Cd2+ derivatives of human carbonic anhydrases I and II and bovine red cell carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) have been prepared. The metal ion in these derivatives is readily displaced by Zn2+. The Cd2+-carbonic anhydrases have appreciable 4-nitrophenyl acetate hydrolase activities. These activities increase with pH as if dependent on the basic form of a group with pKa near 10. The Cd2+-carbonic anhydrases also have significant CO2 hydration activities. The Cd2+ derivatives are strongly inhibited by monovalent anions. In particular, I- is a much more potent inhibitor of the Cd2+ enzymes than of the native enzymes. Acetazolamide (5-acetylamido-1,3,4-thiadiazole 2-sulfonamide) is also a strong inhibitor although its affinity for the Cd2+ enzyme is less than its affinity for the native enzyme.

Topics: Acetazolamide; Animals; Anions; Apoenzymes; Cadmium; Carbon Dioxide; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Cattle; Humans; Hydrogen-Ion Concentration; Iodides; Isoenzymes; Nitrophenols; Zinc

The hydrolysis of 4-nitrophenyl acetate by metal complexes Co(en)2(imH)H2O3+, Co(en)2(bzmH)H2O3+, and Co(en)2(imCH3)H2O3+ (imH = imidazole, bzmH = benzimodazole, imCH3 = methyl imidazole) has been investigated in the pH range 5.4-8.9. The small difference in nucleophilic reactivity in the pH range 5.4-6.7 is assumed to be due to hydrogen bonding abilities of the imidazole and substituted imidazole ligands and small pKa differences (k2(imH) = 2.2 X 10(-2) M-1 sec-1, k2(bzmH) = 5.68 X 10(-2) M-1 sec-1, k2(imCH3) = 1.35 X 10(-2) M-1 sec-1, 40 degrees C, 1 = 0.3 NaClO4, pKa(imH) = 6.2, pKa(imCH3) = 6.2 and pKa(bzmH) = 5.9). In the pH range 7.8-8.9, the differences in nucleophilic reactivity (k3(imH) = 85.5 X 10(-2) M-1 sec-1, k3(bzmH) = 33.4 X 10(-2) M-1 sec-1, 40 degrees C, I = 0.3 NaClO4) are reconciled with a significant steric factor outweighing the acidity of the benzimidazole complex. In the pH region 6.7-7.7, the deviation from linearity is presumably due to both hydroxo and imido ligands functioning as nucleophiles, the latter being about 40 times stronger than the former.

Topics: Allylamine; Amines; Animals; Carbonic Anhydrases; Cattle; Hydrogen-Ion Concentration; Kinetics; Models, Biological; Nitrophenols; Spectrophotometry

Hepatic microsomal carboxylesterase (E.C. 3.1.1.1) from rat liver microsomes showed a different capacity for the hydrolysis of various substrates. In castrated male rats, the enzyme activities towards p-nitrophenylacetate and malathion were decreased. When testosterone propionate was administered to castrated male rats, the activities of p-nitrophenylacetate and malathion hydrolases were reversely increased. However, in ovariectomized female rats, the carboxylesterase activities showed substrate-dependent changes, i.e., increase in p-nitrophenylacetate and malathion hydrolases and decrease in acetanilide and isocarboxazid hydrolases. When estradiol benzoate was administered to ovariectomized female rats, the activities of p-nitrophenyl-acetate and malathion hydrolases were decreased; and acetanilide and isocarboxazid hydrolases were increased. These results suggest that hepatic microsomal carboxylesterases may be, at least in part, regulated by gonadal hormones which exert different effects on the several isozymes of carboxylesterases.

Topics: Acetanilides; Animals; Carboxylic Ester Hydrolases; Castration; Estradiol; Female; Gonadal Steroid Hormones; Hydrolysis; Malathion; Male; Microsomes, Liver; Nitrophenols; Rats; Rats, Inbred Strains; Sex Factors; Testosterone

Stimulation of human milk lipase by deoxycholate and its taurine and glycine conjugates was demonstrated by measuring the esterolysis reaction of 4-nitrophenylacetate. The steroidal surfactants did not bind strongly to the polar substrate but they did bind effectively to a hydrophobic site on the enzyme and these bile salt-enzyme complexes were effective catalysts. These results are compared with those for stimulation of the enzyme by cholate surfactants and it has been demonstrated that the absence of a 7 alpha-OH substituent on the steroid nucleus does not prevent stimulation of either the esterolytic or lipolytic activity of the enzyme.

Topics: Deoxycholic Acid; Enzyme Activation; Glycodeoxycholic Acid; Humans; Hydrolysis; Lipase; Milk, Human; Nitrophenols; Taurodeoxycholic Acid

The pseudo-first-order rate constants of hydrolysis of p-nitrophenylacetate, catalyzed by human milk lipase, have been measured in solutions of 0.01 mol dm-3 Bistris(2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3 -diol) buffer at 310.5 K, containing a range of concentrations of sodium taurocholate and sodium cholate, at pH 8.00 and of sodium cholate at pH 6.5. The effect of pH on the activity of the enzyme has been investigated and the stimulation factors of taurocholate and cholate ions and of cholic acid have been calculated to be equal to 5.3, 3.7 and 10.7, respectively. The essential residues for catalytic activity of the enzyme have ionization constants equal to 6.45-6.46 for pK1 and 8.33-8.40 for pK2. The former value is attributed to the presence of a histidine imidazolium group but the identity of the residue leading to pK2 is not proven.

Topics: Catalysis; Cholic Acid; Cholic Acids; Enzyme Activation; Humans; Hydrogen-Ion Concentration; Hydrolysis; Ions; Kinetics; Lipase; Milk, Human; Nitrophenols; Osmolar Concentration; Taurocholic Acid

Topics: 2-Acetylaminofluorene; Acetanilides; Amidohydrolases; Animals; Aspirin; Benzo(a)pyrene; Benzopyrenes; Carboxylic Ester Hydrolases; Female; Methylcholanthrene; Microsomes, Liver; Nitrophenols; Phenobarbital; Procaine; Rats; Tissue Distribution

The catalytic activities of human red cell carbonic anhydrase (EC 4.2.1.1) isozymes B and C for the hydrolysis of 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone have been compared with their activities towards three other substrates. The substrate specificity (measured as kcat/Km) for either isozyme decreases in this order: CO2 greater than 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone greater than acetaldehyde greater than p-nitrophenyl acetate. Unlike CO2 hydration, enzyme B is slightly more active towards sultone hydrolysis than C. Despite these widely differing activities of both isozymes with regard to different substrates, the inhibition constants for anion and sulfonamide inhibitors are nearly independent of the substrate used. This suggests that the binding sites of these substrates in the enzyme are the same or nearly the same. Earlier studies on 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone from this and other laboratories had underestimated both the intrinsic activity and the susceptibility to anion inhibition of human carbonic anhydrase B. We now find that this was due to the use of acetonitrile as the substrate solvent, which is often contaminated with cyanide, a powerful inhibitor of carbonic anhydrase. The inhibition of human carbonic anhydrase B by several industrial batches of acetonitrile agrees completely with the spectrophotometrically determined cyanide content of these batches.

Topics: Acetaldehyde; Acetonitriles; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Cyanides; Humans; Hydrolysis; Isoenzymes; Kinetics; Nitrophenols; Tosyl Compounds

Fractionation of pancreatic juice by heparin-Sepharose and cholate-Sepharose affinity chromatography indicated that pancreatic carboxylesterase can be separated from pancreatic lipase with the former retained and the latter unretained by both columns. The chromatographic behavior of pancreatic carboxylesterase was found to be similar to that of human milk bile salt-activated lipase. The partially purified pancreatic carboxylesterase had a specific activity of 30 mumol/min per mg protein when assayed with p-nitrophenyl acetate. The reaction mechanism of human pancreatic carboxylesterase was studied using p-nitrophenyl acetate as substrate and taurocholate as activator. The reaction of the enzyme was found to follow a rapid-equilibrium random mechanism. Because of the presence of basal activity, the role of taurocholate can be considered as a non-essential activator and the dissociation constant for the enzyme-taurocholate binary complex was determined to be 0.20 mM. The activation effect of taurocholate consists in increasing the affinity of the enzyme to the substrate (5.6-fold) and in increasing the Vmax (2.3-fold). Based on the kinetic property of human pancreatic carboxylesterase and human milk bile salt-activated lipase with p-nitrophenyl acetate, cholesterol oleate and triolein as substrate, we conclude that they share common substrate specificity but show minor differences in kinetic parameters. Fluorescence studies indicated that both enzymes showed a decreased intrinsic tryptophanyl fluorescence upon incubation with taurocholate. This indicates that bile salt caused a conformational change of the enzymes, with a resultant decreased hydrophobicity in the microenvironment of tryptophan residues.

Topics: Animals; Bile Acids and Salts; Carboxylesterase; Carboxylic Ester Hydrolases; Chromatography, Affinity; Enzyme Activation; Humans; Immunochemistry; Kinetics; Lipase; Middle Aged; Milk; Nitrophenols; Pancreatic Juice; Substrate Specificity

Topics: Acetates; Cetrimonium; Cetrimonium Compounds; Hydrolysis; Kinetics; Micelles; Nitrophenols; Phosphatidylcholines; Polidocanol; Polyethylene Glycols; Surface-Active Agents

First-order rate constants (kL) for hydrolysis of p-nitrophenyl acetate, cationic cyclocytidine, and anionic indomethacin in the presence of buffered liposomal suspensions of positive, negative, and neutral charge were compared to those determined in the corresponding buffers (kB) using the ratio, Rk = kL/kB. Association between the reactants and the liposomes was evaluated by comparing assays for concentration in the filtrates (CF) with the total concentration in the liposomal suspension (CT) using RC = CF/CT. Liposomes did not influence cyclocytidine hydrolysis rates and no association was observed (Rk congruent to RC congruent to 1). In contrast, indomethacin showed approximately 80% reduction in hydrolysis rate and approximately 80% liposome association value (Rk congruent to 0.2 congruent to RC). In neutral and negatively charged liposomal suspensions, p-nitrophenyl acetate displayed approximately 30% decrease in kB (Rk congruent to 0.7) together with approximately 90% liposomal association (RC congruent to 0.1). However, hydrolysis was greatly accelerated in positively charged liposomal suspensions. Loss was described by a biexponential equation where alpha is the fast and beta is the slow pre-exponential coefficient and alpha/beta/kB = 39:6:1. The observed relationships between hydrolysis rates and reactant-liposome associations are reconciled in terms of the hydrophilicity of the reactants and the potential influence of the liposomes on the expected transition states for the hydrolysis reactions.

Topics: Ancitabine; Buffers; Cytarabine; Drug Stability; Hydrolysis; Indomethacin; Kinetics; Liposomes; Models, Chemical; Nitrophenols; Suspensions

Examination of beta-carbons coordinates of seryl, aspartyl and histidyl residues in active sites of alpha-chymotrypsin and subtilisin BPN' shows that a close geometrical arrangement can be obtained in an antiparellel beta-structure. Therefore some polypeptides incorporating serine, aspartic acid and histidine, poly (Gly-Ser-Asp-His-Ala-Pro) and poly [(Asp-Leu-AsP-Leu)10, (His-Leu-Ser-Leu)1], and expected to have some tendency to give rise to an antiparallel beta-conformation, have been prepared and studied. The second polymer only adopts a fairly well-defined beta-structure in aqueous solution. Catalytic activities of these products towards p-nitrophenyl acetate are not improved as compared to histidine. However, kinetic pK of histidine side-chain depends markedly upon the nature of the product, owing probably to a hydrophobic environment effect.

Topics: Amino Acid Sequence; Aspartic Acid; Binding Sites; Chymotrypsin; Circular Dichroism; Histidine; Nitrophenols; Peptides; Protein Conformation; Serine; Spectrophotometry, Infrared; Structure-Activity Relationship; Subtilisins

Bovine milk lipoprotein lipase (LpL) catalyzes the hydrolysis of the water-soluble esters p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB). The same protein and same active site are involved in hydrolysis of water-soluble p-nitrophenyl esters and emulsified trioleoylglycerol since (a) trioleoylglycerol hydrolysis and PNPB hydrolysis activities coelute from the heparin-Sepharose affinity column used to purify LpL and (b) LpL-catalyzed hydrolyses of trioleoylglycerol and PNPB are inhibited to equal extents by phenylmethanesulfonyl fluoride. The effect of apolipoprotein C-II (apoC-II) on the LpL-catalyzed hydrolysis of PNPA and PNPB has been determined. ApoC-II inhibits hydrolysis of both esters, with a maximum extent of inhibition of 70-90%. Inhibition of the LpL-catalyzed hydrolysis of PNPB is specific for apoC-II, since apolipoproteins A-I, C-I, and C-III-2 have little effect on this reaction, and is partial noncompetitive in form. KI values for apoC-II inhibition of the LpL-catalyzed hydrolysis of PNPA and PNPB are in the range 0.26-0.83 microM. The effect of apoC-II on the temperature dependences of LpL-catalyzed hydrolysis of both esters and on NaCl inhibition of LpL-catalyzed PNPB hydrolysis is consistent with a change in rate-determining step with LpL and apoC-II interact. These results indicate not only that there is an interaction between apoC-II and LpL in aqueous solution in the absence of a lipid interface but also that this interaction conformationally modulates the active site of the enzyme.

Topics: Animals; Apolipoprotein C-II; Apolipoproteins; Apolipoproteins C; Cattle; Hydrolysis; Lipoprotein Lipase; Mathematics; Nitrophenols; Time Factors

Topics: Acetates; Amidohydrolases; Animals; Butyrates; Carboxylesterase; Carboxylic Ester Hydrolases; Chemical Precipitation; Chromatography, Gel; Isoelectric Focusing; Isoenzymes; Male; Methods; Microsomes, Liver; Nitrophenols; Rats; Rats, Inbred Strains; Spectrophotometry; Substrate Specificity

Topics: Acetates; Hydrolysis; Kinetics; Liposomes; Nitrophenols

An aldehyde dehydrogenase from rabbit liver, a homogeneous protein on three distinct polyacrylamide-gel systems, has an associated 4-nitrophenyl esterase activity. At pH 7.0 in the presence of 80 micrometer-NADH and 800 micrometer-4-nitrophenyl acetate the enzyme produces NAD+ and a stoicheiometric amount of an aldehyde, as well as hydrolysing the ester. On this and other evidence it is proposed that ester hydrolysis occurs at the usual active site of the enzyme.

Topics: Acetates; Aldehyde Oxidoreductases; Binding Sites; Esterases; Hydrolysis; Iodoacetates; NAD; Nitrophenols

The hydrolysis of 4-nitrophenyl acetate catalysed by cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by steady-state and transient kinetic techniques. NAD+ and NADH stimulated the steady-state rate of ester hydrolysis at concentrations expected on the basis of their Michaelis constants from the dehydrogenase reaction. At higher concentrations of the coenzymes, both NAD+ and NADH inhibited the reaction competitively with respect to 4-nitrophenyl acetate, with inhibition constants of 104 and 197 micron respectively. Propionaldehyde and chloral hydrate are competitive inhibitors of the esterase reaction. A burst in the production of 4-nitrophenoxide ion was observed, with a rate constant of 12 +/- 2s-1 and a burst amplitude that was 30% of that expected on the basis of the known NADH-binding site concentration. The rate-limiting step for the esterase reaction occurs after the formation of 4-nitrophenoxide ion. Arguments are presented for the existence of distinct ester- and aldehyde-binding sites.

Topics: Acetates; Aldehyde Oxidoreductases; Aldehydes; Animals; Chloral Hydrate; Cytoplasm; Kinetics; Liver; NAD; Nitrophenols; Sheep

1. The action of a highly purified phospholipase B from Penicillium notatum on 1-O-alk-1'-enyl-2-acyl-, 1-O-alkyl-2-acyl-, 1,2-diacyl-, 1-acyl- and 2-acyl-sn-glycero-3-phosphocholine, monoacyl-, diacyl- and triacylglycerols, cholesteryl oleate and p-nitrophenyl acetate was studied. 2. The hydrolysis products of the monoethermonoacylglycerophospholipids were identified as fatty acids, 1-O-alk-1'-enyl-sn-glycero-3-phosphocholine and 1-O-alkyl-sn-glycero-3-phosphocholine. The hydrolysis rates were in the following order: 1,2-diacyl-sn-glycero-3-phosphocholine greater than 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphocholine greater than 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine. 3. 1-Acyl-sn-glycero-3-phosphocholine was hydrolyzed about 15 times faster than 2-acyl-sn-glycero-3-phosphocholine. 4. Monoacylglycerols were hydrolyzed at the optimal pH 4.0, but diacyl- and triacylglycerols were not hydrolyzed at various pH values between 4.0 and 9.0. 5. Cholesteryl oleate and p-nitrophenyl acetate were not hydrolyzed.

Topics: Acetates; Cholesterol Esters; Glycerides; Glycerophosphates; Isoelectric Focusing; Nitrophenols; Penicillium; Phosphatidylcholines; Phospholipases; Polyethylene Glycols; Substrate Specificity

Topics: Acylation; Catalysis; Glyceraldehyde-3-Phosphate Dehydrogenases; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Nitrophenols

Topics: Chemical Phenomena; Chemistry; Glutamates; Glutamic Acid; Histidine; Hydrolysis; Nitrophenols; Peptides; Polymers; Research

Topics: Acetates; Acetic Acid; Chemical Phenomena; Chemistry; Nitrophenols; Pyridines; Research; Solvents

1. The reaction of alpha-chymotrypsin with sodium periodate at pH5.0 has been investigated. The enzyme consumes 2 moles of periodate/mole, and there is a concomitant fall in enzymic activity (with respect to l-tyrosine ethyl ester) to 55% of that of the native enzyme. After 3hr. no further change is observed in periodate uptake or in catalytic activity. 2. The oxidized enzyme is a homogeneous preparation of partially active chymotrypsin. 3. In the oxidized enzyme, one of the two methionine residues in the molecule has been converted into its sulphoxide. It is this reaction only that is responsible for the loss of activity. 4. The rate constants for the enzyme-catalysed acylation and deacylation reactions are unaltered by oxidation of the enzyme, both for a non-specific substrate (p-nitrophenyl acetate), and for three specific substrates: N-acetyl-l-tryptophan ethyl ester, N-acetyl-l-tryptophanamide and N-acetyl-l-valine ethyl ester. 5. The K(m) values for the aromatic substrates with the oxidized enzyme are twice those with the native enzyme. No change in Michaelis constant is seen for the non-aromatic substrate N-acetyl-l-valine ethyl ester. 6. The evidence points to the oxidized methionine residue in the modified enzyme being situated in the locus of the active site at which aromatic (or bulky) side chains of the substrates are bound.

Topics: Acylation; Amides; Amino Acids; Biochemical Phenomena; Biochemistry; Catalysis; Chymotrypsin; Imidazoles; Kinetics; Methionine; Nitrophenols; Periodic Acid; Research; Spectrophotometry; Tryptophan; Tyrosine; Ultracentrifugation; Valine

Topics: Acetates; Carbon Isotopes; Chemistry, Pharmaceutical; Kinetics; Nitrobenzenes; Nitrophenols; Research

Topics: Acetates; Catalysis; Cysteine; Glutathione; Homocysteine; Kinetics; Mercaptoethylamines; Nitrophenols; Research; Spectrophotometry; Sulfhydryl Compounds

Topics: Glyceraldehyde 3-Phosphate; Glyceraldehyde-3-Phosphate Dehydrogenases; Hydrolysis; Nitrophenols; Oxidoreductases; Saccharomyces cerevisiae; Yeasts

Topics: Acetates; Imidazoles; Nitrophenols; Peptides

Topics: Copper; Glycine; Ions; Nitrophenols; Peptides; Phenylacetates

Topics: Acetylation; Chemical Phenomena; Chemistry; Glyceraldehyde; Glyceraldehyde-3-Phosphate Dehydrogenases; Nitrophenols; Oxidoreductases; Phenylacetates; Phosphates; Research

Topics: Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Coenzymes; Creatine; Cysteine; Enzyme Inhibitors; Ions; Manganese; Nitrophenols; Phenylacetates; Phosphotransferases; Polyphosphates; Research; Spectrophotometry; Sulfhydryl Compounds

Topics: Animals; Cattle; Erythrocytes; Hydro-Lyases; Nitrophenols; Phenylacetates; Research

Topics: Animals; Hemoglobins; Hydrogen; Metmyoglobin; Nitrophenols; Phenylacetates; Protons; Semen; Sperm Whale

Topics: Acetates; Chymotrypsin; Hydrolysis; Kinetics; Nitrophenols

Topics: Acetates; Glyceraldehyde 3-Phosphate; Hydrolysis; Nitrophenols; Oxidoreductases

Topics: Acetates; Acetylation; Alcohols; Butanols; Chymotrypsin; Nitrophenols

Topics: Acetates; Histidine; Hydrolysis; Methylhistidines; Nitrophenols; Peptides

Topics: Catalysis; Chymotrypsin; Hydrolysis; Nitrophenols; Phenylacetates

Topics: Catalysis; Hydrolysis; Nitrophenols

Topics: Acetates; Butyrates; Esterases; Humans; Lipid Metabolism; Nitrophenols; Propionates