(S)-Mandelate Dehydrogenase from Pseudomonas putida: Mechanistic Studies with Alternate Substrates and pH and Kinetic Isotope Effects

(S)-Mandelate dehydrogenase from Pseudomonas putida, a member of the flavin mononucleotide-dependent α-hydroxy acid oxidase/dehydrogenase family, oxidizes (S)-mandelate to benzoylformate. The enzyme was purified with a carboxy-terminal histidine tag. Steady-state kinetic parameters indicate that it preferentially binds large substrates. A good correlation was obtained between the k cat, the substrate kinetic isotope effect (KIE), and the pK a of the substrate α-proton. The k cat decreased and the KIE increased for substrates whose α-protons have pK as higher than that of mandelate. These results support a mechanism involving a carbanion intermediate but are difficult to reconcile with one involving a direct hydride transfer. pH effects on steady-state parameters were determined with (S)-mandelate and a slow substrate, (R,S)-3-phenyllactate. The k cat/K m pH profile shows that two groups with apparent pK as of 5.5 and 8.9 in the free enzyme are important for activity. These pK as are shifted to 5.1 and 9.6 on binding (S)-mandelate, as shown in the k cat pH profile. The pH dependence of the KIEs suggests that the residues with these pK as are involved in the α-carbon−hydrogen bond-breaking step. pH dependencies of the inhibition constants for competitive inhibitors identified these residues as histidine 274 and arginine 277. We propose that histidine 274 is the base that abstracts the substrate α-proton and arginine 277 is important for substrate binding as well as stabilization of the carbanion/enolate intermediate.