Co-solvent effects on reaction rate and reaction equilibrium of an enzymatic peptide hydrolysis

This work presents an approach that expresses the Michaelis constant K-M(a) and the equilibrium constant K-th of an enzymatic peptide hydrolysis based on thermodynamic activities instead of concentrations. This provides K-M(a) and K-th 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 alpha-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 (K-M) and equilibrium constant (K-x) 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 K-M(a) and K-th for the co-solvent free reaction. Based on these constants, the co-solvent effect on K-M and K-x 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.