nitrophenols and 4-nitrophenyl-palmitate

The paper at hand deals with the influence of the pH-value on the conformation and activity of the lipase B from Candida antarctica (CalB) which is incorporated in a bicontinuous microemulsion. The microemulsion used for this purpose consists of water/NaCl, n-octane, and the non-ionic surfactant penthaethylene glycol monodecylether (C10E5). The conformational study clearly shows (1) that CalB molecules are partitioned between the interfacial monolayer and the water domains and (2) that the pH-value of the microemulsion's water domains strongly influences the conformation of CalB at the interfacial monolayer. From these observations we conclude that there is a continuous exchange between the CalB molecules, which are located at the interfacial monolayer and those which are located in the water domains of the microemulsion. This exchange strongly influences the CalB conformation in both regions. In addition to the conformation, we also studied the catalytic activity of CalB. The catalytic measurements revealed a bell-shaped dependence between the CalB activity and the pH-value. The maximum catalytic activity of CalB in bicontinuous microemulsions was observed at pH=5.5. At this pH we observed the highest amount of α-helix conformation of the CalB molecules that are located at the interfacial monolayer, which, in turn, allows connecting the activity with the conformation.

Topics: Biocatalysis; Candida; Circular Dichroism; Emulsions; Fungal Proteins; Hydrogen-Ion Concentration; Kinetics; Lipase; Models, Chemical; Molecular Structure; Nitrophenols; Octanes; Palmitates; Palmitic Acid; Protein Conformation; Sodium Chloride; Surface Properties; Surface-Active Agents; Water

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

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

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

(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

The fatty acyl (lipid) p-nitrophenyl esters p-nitrophenyl caprylate, p-nitrophenyl laurate and p-nitrophenyl palmitate that are incorporated at a few mol % into mixed micelles with Triton X-100 are substrates for bovine milk lipoprotein lipase. When the concentration of components of the mixed micelles is approximately equal to or greater than the critical micelle concentration, time courses for lipoprotein lipase-catalyzed hydrolysis of the esters are described by the integrated form of the Michaelis-Menten equation. Least square fitting to the integrated equation therefore allows calculation of the interfacial kinetic parameters Km and Vmax from single runs. The computational methodology used to determine the interfacial kinetic parameters is described in this paper and is used to determine the intrinsic substrate fatty acyl specificity of lipoprotein lipase catalysis, which is reflected in the magnitude of kcat/Km and kcat. The results for interfacial lipoprotein lipase catalysis, along with previously determined kinetic parameters for the water-soluble esters p-nitrophenyl acetate and p-nitrophenyl butyrate, indicate that lipoprotein lipase has highest specificity for the substrates that have fatty acyl chains of intermediate length (i.e. p-nitrophenyl butyrate and p-nitrophenyl caprylate). The fatty acid products do not cause product inhibition during lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles. The effects of the nucleophiles hydroxylamine, hydrazine, and ethylenediamine on Km and Vmax for lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl laurate are consistent with trapping of a lauryl-lipoprotein lipase intermediate. This mechanism is confirmed by analysis of the product lauryl hydroxamate when hydroxylamine is the nucleophile. Hence, lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles occurs via an interfacial acyl-lipoprotein lipase mechanism that is rate-limited by hydrolysis of the acyl-enzyme intermediate.

Topics: Animals; Caprylates; Cattle; Female; Kinetics; Laurates; Lipid Metabolism; Lipoprotein Lipase; Mathematics; Micelles; Milk; Nitrophenols; Palmitates