Role of arginine 277 in (S)-mandelate dehydrogenase from Pseudomonas putida in substrate binding and transition state stabilization.
(S)-Mandelate dehydrogenase from Pseudomonas putida is an FMN-dependent alpha-hydroxy acid dehydrogenase. Structural studies of two homologous enzymes, glycolate oxidase and flavocytochrome b(2), indicated that a conserved arginine residue (R277 in MDH) interacts with the product carboxylate group [Lindqvist, Y., Branden, C.-I., Mathews, F. S., and Lederer, F. (1991) J. Biol. Chem. 266, 3198-3207]. The catalytic role of R277 was investigated by site-specific mutagenesis together with chemical rescue experiments. The R277K, R277G, R277H, and R277L proteins were generated and purified in active forms. The k(cat) for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its K(m) value was 40-fold lower; in contrast, k(cat)s for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and K(m) values were 5-15-fold lower compared to R277K. The K(d)s for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. The k(cat) for R277G could be enhanced by the addition of exogenous guanidines or imidazoles; the maximum rescued k(cat) was approximately 70% of the wt-MDH value. Only reagents that were positively charged and could function as hydrogen bond donors were effective rescue agents. Our results indicate that R277 plays a major role in transition state stabilization through its positive charge-consistent with a mechanism involving a carbanion intermediate. The positive charge has a relatively small contribution toward substrate binding. R277 also forms a specific hydrogen bond with both the substrate and the transition state; this interaction contributes significantly to the low K(m) for (S)-mandelate.
Lehoux
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Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201, USA.
Mitra
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B
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001120:Arginine; D020134:Catalytic Domain; D017124:Conserved Sequence; D005486:Flavin Mononucleotide; D005420:Flavoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006146:Guanidines / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013816:Thermodynamics
D005420:Flavoproteins; D006146:Guanidines; D007093:Imidazoles; D008024:Ligands; D011994:Recombinant Proteins; D005486:Flavin Mononucleotide; D001120:Arginine; D000429:Alcohol Oxidoreductases; C021538:L-mandelate dehydrogenase