Abstract
Flavocytochrome b2 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 DVmax and D(V/Klactate) values are both 3.0 with the wild-type enzyme at pH 7.5 and 25 °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 D20Vmax 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/Klactate) values are 0.9, 0.44, and 1.0, respectively. The Dkred 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 Vmax values for the wild-type enzyme and the flavin domain are 32 and 15% limited by viscosity, respectively. In contrast, the V/Klactate 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.
Recommended Citation
P. Sobrado et al., "Probing the relative timing of hydrogen abstraction steps in the flavocytochrome b2 reaction with primary and solvent deuterium isotope effects and mutant enzymes," Biochemistry, vol. 40, no. 4, pp. 994 - 1001, American Chemical Society, Jan 2001.
The definitive version is available at https://doi.org/10.1021/bi002283d
Department(s)
Chemistry
International Standard Serial Number (ISSN)
0006-2960
Document Type
Article - Journal
Document Version
Citation
File Type
text
Language(s)
English
Rights
© 2024 American Chemical Society, All rights reserved.
Publication Date
30 Jan 2001
PubMed ID
11170421
