Co-solvents are known to influence the Michaelis constant K M of enzyme-catalyzed reactions. In the literature, co-solvent effects on K M 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 K M a . 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 K M of ACP and decreased K M of nicotinamide adenine dinucleotide (NADH). To predict K M a 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 K M values, their activity-based pendants K M a 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 K M of 2-pentanone and K M of NADH. Based on K M a values obtained from K M in co-solvent-free conditions and activity coefficients from ePC-SAFT, the influence of the co-solvent on K M 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.

Dominik Böttcher

Publications