(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 alpha-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 alpha-proton. The k(cat) decreased and the KIE increased for substrates whose alpha-protons have pK(a)s 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(a)s of 5.5 and 8.9 in the free enzyme are important for activity. These pK(a)s 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(a)s are involved in the alpha-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 alpha-proton and arginine 277 is important for substrate binding as well as stabilization of the carbanion/enolate intermediate.