Solvent Isotope Effects in Reactions Catalyzed by Lactate Dehydrogenase and Phenylalanine Dehydrogenase

Background/Objectives: L-Lactate dehydrogenase (LDH) and L-phenylalanine dehydrogenase (PheDH) are enzymes involved in important metabolic pathways and may be affected by structural modifications of their substrates. This study aimed to investigate the effects of selected substrate modifications on LDH and PheDH activity. Methods: The solvent isotope effect (SIE) method was used to investigate the mechanism of enzyme action. Numerical values of kinetic parameters were determined by means of the non-competitive spectrophotometric method, and SIEs were calculated for the Vmax and Vmax/Km parameters. Results: Both enzymes showed reduced activity toward modified substrates compared with their native substrates. For LDH, the lowest Vmax was observed for 3-bromopyruvic acid, while phenylpyruvic acid and 4′-hydroxyphenylpyruvic acid also showed decreased activity, indicating an important role of steric hindrance in substrate binding. PheDH activity decreased with both halogenated L-phenylalanine derivatives, with the chlorinated compound showing the strongest reduction. High SIE values indicated significant solvent involvement in substrate binding and catalytic turnover. In PheDH-catalyzed reactions, the isotope effects were consistent with water-mediated proton transfer. Conclusions: Halogenation and hydroxylation significantly affect the substrate recognition and catalytic activity of LDH and PheDH. The results demonstrate that steric and electronic effects, as well as solvent-mediated proton transfer, contribute to the catalytic mechanisms of both enzymes.