alpha-chymotrypsin and trimethyloxamine

Trimethylamine N-Oxide (TMAO) is an important metabolite, which is derived from choline, betaine, and carnitine in various organisms. In humans, it is synthesized through gut microbiota and is abundantly found in serum and cerebrospinal fluid (CSF). Although TMAO is a stress protectant especially in urea-rich organisms, it is an atherogenic agent in humans and is associated with various diseases. Studies have also unveiled its exceptional role in protein folding and restoration of mutant protein functions. However, most of these data were obtained from studies carried on fast-folding proteins. In the present study, we have investigated the effect of TMAO on the folding behavior of a well-characterized protein with slow folding kinetics, carbonic anhydrase (CA). We discovered that TMAO inhibits the folding of this protein via its effect on proline cis-trans isomerization. Furthermore, TMAO is capable of inducing cell cycle arrest. This study highlights the potential role of TMAO in developing proteopathies and associated diseases.

Topics: Calorimetry; Carbonic Anhydrases; Cell Cycle Checkpoints; Cell Survival; Chymotrypsin; Gastrointestinal Tract; HeLa Cells; Horseradish Peroxidase; Humans; Isomerism; Kinetics; Methylamines; Protein Aggregates; Protein Conformation; Protein Folding; Protein Refolding; Protein Stability; Thermodynamics

The application of co-solvents and high pressure has been reported to be an efficient means to tune the kinetics of enzyme-catalyzed reactions. Co-solvents and pressure can lead to increased reaction rates without sacrificing enzyme stability, while temperature and pH operation windows are generally very narrow. Quantitative prediction of co-solvent and pressure effects on enzymatic reactions has not been successfully addressed in the literature. Herein, we are introducing a thermodynamic approach that is based on molecular interactions in the form of activity coefficients of substrate and of enzyme in the multi-component solution. This allowed us to quantitatively predict the combined effect of co-solvent and pressure on the kinetic constants, i.e. the Michaelis constant K

Topics: Chymotrypsin; Dimethyl Sulfoxide; Hydrolysis; Kinetics; Methylamines; Phenylalanine; Pressure; Solvents; Thermodynamics; Urea; Water

High pressure is an important feature of certain natural environments, such as the deep sea where pressures up to about 1000 bar are encountered. Further, pressure effects on biosystems are of increasing interest for biotechnological applications, such as baroenzymology. We studied the effect of two different natural osmolyte mixtures, with major components being glycine and trimethylamine-N-oxide (TMAO), on the activity of α-chymotrypsin, using high-pressure stopped-flow methodology in combination with fast UV/Vis detection. We show that pressure is not only able to drastically enhance the catalytic activity and efficiency of the enzyme, but also that glycine has a significant and diverse effect on the enzymatic activity and volumetric properties of the reaction compared to TMAO. The results might not only help to understand the modulation of enzymatic reactions by natural osmolytes, but also elucidate ways to optimize enzymatic processes in biotechnological applications.

Topics: Chymotrypsin; Glycine; Hydrolysis; Kinetics; Methylamines; Osmolar Concentration; Pressure; Substrate Specificity; Thermodynamics

This work presents an approach that expresses the Michaelis constant KaM and the equilibrium constant Kth of an enzymatic peptide hydrolysis based on thermodynamic activities instead of concentrations. This provides KaM and Kth values that are independent of any co-solvent. To this end, the hydrolysis reaction of N-succinyl-l-phenylalanine-p-nitroanilide catalysed by the enzyme α-chymotrypsin was studied in pure buffer and in the presence of the co-solvents dimethyl sulfoxide, trimethylamine-N-oxide, urea, and two salts. A strong influence of the co-solvents on the measured Michaelis constant (KM) and equilibrium constant (Kx) was observed, which was found to be caused by molecular interactions expressed as activity coefficients. Substrate and product activity coefficients were used to calculate the activity-based values KaM and Kth for the co-solvent free reaction. Based on these constants, the co-solvent effect on KM and Kx was predicted in almost quantitative agreement with the experimental data. The approach presented here does not only reveal the importance of understanding the thermodynamic non-ideality of reactions taking place in biological solutions and in many technological applications, it also provides a framework for interpreting and quantifying the multifaceted co-solvent effects on enzyme-catalysed reactions that are known and have been observed experimentally for a long time.

Topics: Animals; Calcium Chloride; Cattle; Chymotrypsin; Dimethyl Sulfoxide; Hydrolysis; Kinetics; Methylamines; Phenylalanine; Sodium Chloride; Solvents; Thermodynamics; Urea; Water

We have studied the effect of guanidinium chloride (Gdn.Cl) and different osmolytes such as betaine, trimethylamine-N-oxide (TMAO) and urea on the rate of chymotrypsin catalyzed reaction. The rates were measured using three synthetic chromogenic substrates, succinyl-ala-ala-pro-arg-pNA (AAPR), succinyl-ala-ala-pro-leu-pNA (AAPL), and succinyl-ala-ala-pro-phe-pNA (AAPF). Qualitatively, the results with the three substrates were identical. Guanidinium chloride and urea produced a linear decrease while TMAO produced a linear increase in the rate with increase in osmolyte concentration. Betaine had practically no effect on the rate of enzyme catalyzed reaction up to a concentration of 1.2 M. However, quantitatively the rate change per molar concentration of osmolyte (or Gdn.Cl) was significantly larger for AAPR that has a polar and cationic reactive site residue than the two substrates (AAPL and AAPF) that have non-polar reactive site residues. These results suggest that the chemical nature of the substrate (and presumably the active site of the enzyme) plays an important role in determining the effect of osmolytes in enzyme catalyzed reactions.

Topics: Animals; Betaine; Biocatalysis; Catalytic Domain; Cattle; Chymotrypsin; Guanidine; Kinetics; Methylamines; Peptides; Urea

We analyzed the effect of a natural osmolyte, trimethylamine N-oxide (TMAO), on structural properties and conformational stabilities of several proteins under macromolecular crowding conditions by a set of biophysical techniques. We also used the solvent interaction analysis method to look at the peculiarities of the TMAO-protein interactions under crowded conditions. To this end, we analyzed the partitioning of these proteins in TMAO-free and TMAO-containing aqueous two-phase systems (ATPSs). These ATPSs had the same polymer composition of 6.0 wt.% PEG-8000 and 12.0 wt.% dextran-75, and same ionic composition of 0.01 M K/NaPB, pH 7.4. These analyses revealed that there is no direct interaction of TMAO with proteins, suggesting that the TMAO effects on the protein structure in crowded solutions occur via the effects of this osmolyte on solvent properties of aqueous media. The effects of TMAO on protein structure in the presence of polymers were rather complex and protein-specific. Curiously, our study revealed that in highly concentrated polymer solutions, TMAO does not always act to promote further protein folding.

Topics: Animals; Calorimetry, Differential Scanning; Cattle; Chymotrypsin; Circular Dichroism; Dextrans; Humans; Hydrogen-Ion Concentration; Light; Methylamines; Pancreas; Polyethylene Glycols; Polymers; Protein Binding; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Scattering, Radiation; Solvents; Spectrometry, Fluorescence; Temperature; Water

We investigated the combined effects of cosolvents and pressure on the hydrolysis of a model peptide catalysed by α-chymotrypsin. The enzymatic activity was measured in the pressure range from 0.1 to 200 MPa using a high-pressure stopped-flow systems with 10 ms time resolution. A kosmotropic (trimethalymine-N-oxide, TMAO) and chaotropic (urea) cosolvent and mixtures thereof were used as cosolvents. High pressure enhances the hydrolysis rate as a consequence of a negative activation volume, ΔV(#), which, depending on the cosolvent system, amounts to -2 to -4 mL mol(-1). A more negative activation volume can be explained by a smaller compression of the ES complex relative to the transition state. Kinetic constants, such as kcat and the Michaelis constant KM, were determined for all solution conditions as a function of pressure. With increasing pressure, kcat increases by about 35% and its pressure dependence by a factor of 1.9 upon addition of 2 M urea, whereas 1 M TMAO has no significant effect on kcat and its pressure dependence. Similarly, KM increases upon addition of urea 6-fold. Addition of TMAO compensates the urea-effect on kcat and KM to some extent. The maximum rate of the enzymatic reaction increases with increasing pressure in all solutions except in the TMAO : urea 1 : 2 mixture, where, remarkably, pressure is found to have no effect on the rate of the enzymatic reaction anymore. Our data clearly show that compatible solutes can easily override deleterious effects of harsh environmental conditions, such as high hydrostatic pressures in the 100 MPa range, which is the maximum pressure encountered in the deep biosphere on Earth.

Topics: Biocatalysis; Chymotrypsin; Enzyme Activation; Hydrolysis; Kinetics; Methylamines; Peptides; Pressure; Solvents; Urea

An assay for the determination of the equilibrium constant for heteroassociation of α-chymotrypsin and soybean trypsin inhibitor via fluorescence depolarization is described. Results obtained at neutral pH in saline buffer were consistent with prior determinations via sedimentation equilibrium and static light scattering. The dependence of the association equilibrium constant upon the concentrations of urea and trimethylamine-N-oxide (TMAO) added individually and in mixtures was determined at several temperatures. It was found that subdenaturing concentrations of urea decrease the extent of heteroassociation and that added TMAO increases the extent of heteroassociation. The effects of both cosolutes in mixtures upon the equilibrium heteroassociation of α-chymotrypsin and soybean trypsin inhibitor appear to be additive. A thermodynamic analysis of the combined results is presented.

Topics: Chymotrypsin; Fluorescence; Glycine max; Hydrogen-Ion Concentration; Methylamines; Thermodynamics; Trypsin Inhibitor, Kunitz Soybean; Urea

Osmolytes form a class of naturally occurring small compounds known to protect proteins in their native folded and functional states. Among the osmolytes, trimethylamine-N-oxide (TMAO) has received special interest lately because it has shown an extraordinary capability to support folding of denatured to native-like species, which show significant functional activity. Most enzymes and/or proteins are commonly stored in glycerol to maintain their activity/function. In the present study, we tested whether TMAO can be a better solute than glycerol for two commonly used proteases, trypsin and chymotrypsin. Our enzyme kinetic data suggest that the enzyme activity of trypsin is significantly enhanced in TMAO compared to glycerol, whereas chymotrypsin activity is not significantly changed in either case. These results are in accordance with the osmolyte effects on the folding of these enzymes, as judged by data from fluorescence emission spectroscopy. These results suggest that TMAO may be a better solute than glycerol to maintain optimal tryptic enzyme activity.

Topics: Catalysis; Chymotrypsin; Enzyme Activation; Glycerol; Kinetics; Methylamines; Osmotic Pressure; Protein Denaturation; Protein Folding; Spectrometry, Fluorescence; Time Factors; Trypsin