Probing the relative timing of hydrogen abstraction steps in the flavocytochrome b(2) reaction with primary and solvent deuterium isotope effects andmutant enzymes

Flavocytochrome b(2) catalyzes the oxidation of lactate to pyruvate. Primar y 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 wi ld-type enzyme, a mutant protein lacking the heme domain, and the D282N enz yme. The V-D(max), and (D)(V/K-lactate) values are both 3.0 with the wild-t ype enzyme at pH 7.5 and 25 degreesC, increasing to about 3.6 with the flav in domain and increasing further to about 4.5 with the D282N enzyme. Under these conditions, the V-D20(max) values are 1.38, 1.18, and 0.98 for the wi ld-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(re d) value is 5.4 for the wild-type enzyme and 3.5 for the flavin domain, whe reas the solvent isotope effect on this kinetic parameter is 1.0 for both e nzymes. 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-lactat e value for the flavin domain increases about 2-fold at moderate concentrat ions of glycerol, The data are consistent with a minimal chemical mechanism in which the lactate hydroxyl proton is not in night in the transition sta te 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 solu tion conditions. Removal of the hydroxyl proton may occur in this pre-equil ibrium.