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

Abstract

This work presents an approach that expresses the Michaelis constant K^a_M and the equilibrium constant K_th of an enzymatic peptide hydrolysis based on thermodynamic activities instead of concentrations. This provides K^a_M 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 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 (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^a_M 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.