title: Arginine 165/arginine 277 pair in (S)-mandelate dehydrogenase from Pseudomonas putida: role in catalysis and substrate binding.
abstract: (S)-Mandelate dehydrogenase from Pseudomonas putida belongs to a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids. Active site structures of three homologous enzymes, including MDH, show the presence of two conserved arginine residues in close juxtaposition (Arg165 and Arg277 in MDH). Arg277 has an important catalytic role; it stabilizes both the ground and transition states through its positive charge as well as a hydrogen bond [Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065]. In this study, we examined the role of Arg165 and the overall importance of the Arg165/Arg277 pair. Single mutants at Arg165 as well as double mutants at Arg165 and Arg277 were characterized. Our results show that Arg165 has a role similar to, but less critical than, that of Arg277. It stabilizes the transition state through its positive charge and the ground state through a charge-independent interaction, most likely, a hydrogen bond. Though the k(cat)s for the charge-conserved mutants, R165K and R277K, were only 3-5-fold lower than those of wild-type MDH (wtMDH), the k(cat) for R165K/R277K was approximately 350-fold lower. Thus, at least one arginine residue is required for the optimal substrate orientation and catalysis. Stopped-flow studies show that the FMN reduction step is completely rate-limiting for both wtMDH and the arginine mutants, with the possible exception of R165E. Substrate isotope effects indicate that the carbon-hydrogen bond-breaking step is only partially rate-limiting for wtMDH but fully rate-limiting for the mutants. pH profiles of R165M conclusively show that the pK(a) of 9.3 in free wtMDH does not belong to Arg165.
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry*; D001667:Binding, Competitive / Q000235:genetics; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D005486:Flavin Mononucleotide / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008333:Mandelic Acids / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010648:Phenylacetates / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013447:Sulfites / Q000378:metabolism
chemical_list: D008024:Ligands; D008333:Mandelic Acids; D010648:Phenylacetates; D013447:Sulfites; D005486:Flavin Mononucleotide; D001120:Arginine; D000429:Alcohol Oxidoreductases; C021538:L-mandelate dehydrogenase; C025136:phenylacetic acid; C037938:mandelic acid