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.

An activity-based approach to predict combined influence of pressure and co-solvent on enzymatic reaction kinetics is presented.

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Co-solvents are known to influence the Michaelis constant of enzyme-catalyzed reactions. In the literature, co-solvent effects on are usually explained by interactions between enzyme and co-solvent. Very recent works replaced substrate concentrations with thermodynamic activities to separate enzyme-co-solvent from substrate-co-solvent interactions This yields the thermodynamic-activity-based Michalis constant . In this work, this approach was extended to alcohol dehydrogenase (ADH)-catalyzed reduction of acetophenone (ACP), a two-substrate reaction. It was experimentally found that polyethylene glycol (PEG) 6000 increased of ACP and decreased of nicotinamide adenine dinucleotide (NADH). To predict values, non-covalent interactions between substrates and reaction media were taken into account by electrolyte perturbed-chain statistical associating fluid theory (ePC-SAFT) modelling. In contrast to experimental values, their activity-based pendants were independent of co-solvent. To further verify the approach, the reduction of 2-pentanone catalyzed by the same ADH was investigated. Interestingly, the addition of PEG caused a decrease of both of 2-pentanone and of NADH. Based on values obtained from in co-solvent-free conditions and activity coefficients from ePC-SAFT, the influence of the co-solvent on was quantitatively predicted. Thus, the approach known for pseudo one-substrate reactions was successfully transferred to two-substrate reactions. Furthermore, the advantage of thermodynamic activities over concentrations in the field of enzyme kinetics is highlighted.