A transient intermediate in the reaction catalyzed by (S)-mandelate dehydrogenase from Pseudomonas putida.
(S)-Mandelate dehydrogenase from Pseudomonas putida is a member of a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids to alpha-ketoacids. The reductive half-reaction consists of the steps involved in substrate oxidation and FMN reduction. In this study, we investigated the mechanism of this half-reaction in detail. At low temperatures, a transient intermediate was formed in the course of the FMN reduction reaction. This intermediate is characteristic of a charge-transfer complex of oxidized FMN and an electron-rich donor and is formed prior to full reduction of the flavin. The intermediate was not due to binding of anionic substrates or inhibitors. It was only observed with efficient substrates that have high k(cat) values. At higher temperatures, it was formed within the dead time of the stopped-flow instrument. The rate of formation of the intermediate was 3-4-fold faster than its rate of disappearance; the former had a larger isotope effect. This suggests that the charge-transfer donor is an electron-rich carbanion/enolate intermediate that is generated by the base-catalyzed abstraction of the substrate alpha-proton. This is consistent with the observation that the intermediate was not observed with the R277K and R277G mutants, which have been shown to destabilize the carbanion intermediate (Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065). Thus, the MDH reaction has two rate-limiting steps of similar activation energies: the formation and breakdown of a distinct intermediate, with the latter step being slightly more rate limiting. We also show that MDH is capable of catalyzing the reverse reaction, the reoxidation of reduced MDH by the product ketoacid, benzoylformate. The transient intermediate was observed during the reverse reaction as well, confirming that it is indeed a true intermediate in the MDH reaction pathway.
Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
Asteriani R
AR
Dewanti
Bharati
B
Mitra
D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D002384:Catalysis; D003903:Deuterium; D005486:Flavin Mononucleotide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D016958:Pseudomonas putida / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics
D005486:Flavin Mononucleotide; D001120:Arginine; D003903:Deuterium; D000429:Alcohol Oxidoreductases; C021538:L-mandelate dehydrogenase