Flavocytochrome b(2) catalyzes the oxidation of lactate to pyruvate. Primary deuterium and solvent kinetic isotope effects have been used to determine the relative timing of cleavage of the lactate O-H and C-H bonds by the wild-type enzyme, a mutant protein lacking the heme domain, and the D282N enzyme. The (D)V(max) and (D)(V/K(lactate)) values are both 3.0 with the wild-type enzyme at pH 7.5 and 25 degrees C, increasing to about 3.6 with the flavin domain and increasing further to about 4.5 with the D282N enzyme. Under these conditions, the (D20)V(max) values are 1.38, 1.18, and 0.98 for the wild-type enzyme, the flavin domain, and the D282N enzyme, respectively; the (D20)(V/K(lactate)) values are 0.9, 0.44, and 1.0, respectively. The (D)k(red) value is 5.4 for the wild-type enzyme and 3.5 for the flavin domain, whereas the solvent isotope effect on this kinetic parameter is 1.0 for both enzymes. The V(max) values for the wild-type enzyme and the flavin domain are 32 and 15% limited by viscosity, respectively. In contrast, the V/K(lactate) value for the flavin domain increases about 2-fold at moderate concentrations of glycerol. The data are consistent with a minimal chemical mechanism in which the lactate hydroxyl proton is not in flight in the transition state for C-H bond cleavage and there is an internal equilibrium involving the lactate-bound enzyme prior to C-H bond cleavage which is sensitive to solution conditions. Removal of the hydroxyl proton may occur in this pre-equilibrium.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|