Prediction and Experimental Validation of Co‐Solvent Influence on Michaelis Constants: A Thermodynamic Activity‐Based Approach

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.