Molecular interpretation of inhibition by excess substrate in flavocytochrome b2: a study with wild-type and Y143F mutant enzymes.

Abstract

The crystal structure of flavocytochrome b2 (L-lactate dehydrogenase) from Saccharomyces cerevisiae suggests that Tyr143 plays a dual role at the active site: it contributes to substrate binding and, most importantly, makes a hydrogen bond to a heme propionate, which could facilitate communication between the domains. Previous work on the Y143F mutant enzyme provided support for these hypotheses [Miles, C. S., Rouvière-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., Black, M. T., & Chapman, S. K. (1992) Biochem. J. 285, 187-192; Rouvière-Fourmy, N., Capeillère-Blandin, C., & Lederer, F. (1994) Biochemistry 33, 798-806]. In the course of kinetic comparisons between the wild-type (WT) enzyme and the Y143F mutant protein, we observed for the latter signs of inhibition by excess substrate at much lower concentrations than observed for the former. A detailed investigation of the phenomenon has shown that, for the wild-type and Y143F forms, lactate at high concentrations inhibits both cytochrome c and ferricyanide reduction. In these cases, inhibition appears to be a specific effect, since acetate at identical concentrations exerts an inhibitory effect that is markedly weaker than that of lactate. In the pre-steady-state, in the absence of acceptor, flavin and heme reduction are unaffected by high substrate concentrations in the WT enzyme case. For the Y143F mutant, flavin reduction is similarly unaffected, but heme reduction is inhibited to nearly the same extent by high lactate and acetate concentrations. In this case, inhibition can probably be ascribed to ionic strength effects. The combination of stopped-flow and steady-state results suggests that lactate binds with weak affinity at the active site when the flavin is in the semiquinone state, preventing electron transfer to heme b2 and hence to acceptors. This phenomenon is analogous to the inhibition exerted by pyruvate when bound to the enzyme at the semiquinone stage [Tegoni, M., Janot, J. M., & Labeyrie, F. (1990) Eur. J. Biochem. 190, 329-342]. We suggest that the substrate carboxylate and the heme propionate of the mobile heme-binding domain compete for the Tyr143 hydroxyl group, hence for approach to the flavin. In the Y143F mutant enzyme, in which the interdomain interaction is impaired, competition would play in favor of the substrate, resulting in the inhibition at lower lactate concentrations than observed for the wild-type enzyme.