Solvent and primary deuterium isotope effects show that lactate CH and OH bond cleavages are concerted in Y254F flavocytochrome b2, consistent with a hydride transfer mechanism.
Yeast flavocytochrome b(2) catalyzes the oxidation of lactate to pyruvate; because of the wealth of structural and mechanistic information available, this enzyme has served as the model for the family of flavoproteins catalyzing oxidation of alpha-hydroxy acids. Primary deuterium and solvent isotope effects have now been used to analyze the effects of mutating the active site residue Tyr254 to phenylalanine. Both the V(max) and the V/K(lactate) values decrease about 40-fold in the mutant enzyme. The primary deuterium isotope effects on the V(max) and the V/K(lactate) values increase to 5.0, equivalent to the intrinsic isotope effect for the wild-type enzyme. In addition, both the V(max) and the V/K(lactate) values exhibit solvent isotope effects of 1.5. Measurement of the solvent isotope effect with deuterated lactate establishes that the primary and solvent isotope effects arise from the same chemical step, consistent with concerted cleavage of the lactate OH and CH bonds. The pH dependence of the mutant enzyme is not significantly different from that of the wild-type enzyme; this is most consistent with a requirement that the side chain of Tyr254 be uncharged for catalysis. The results support a hydride transfer mechanism for the mutant protein and, by extension, wild-type flavocytochrome b(2) and the other flavoproteins catalyzing oxidation of alpha-hydroxy acids.
Sobrado
Pablo
P
Departments of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Fitzpatrick
Paul F
PF
D002384:Catalysis; D003903:Deuterium / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D006859:Hydrogen; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006880:Hydroxy Acids / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D042844:L-Lactate Dehydrogenase (Cytochrome) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019344:Lactic Acid / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D012997:Solvents; D014443:Tyrosine / Q000235:genetics
D006880:Hydroxy Acids; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents; D019344:Lactic Acid; D014443:Tyrosine; D010649:Phenylalanine; D006859:Hydrogen; D003903:Deuterium; D042844:L-Lactate Dehydrogenase (Cytochrome)