MOLECULAR INTERPRETATION OF INHIBITION BY EXCESS SUBSTRATE IN FLAVOCYTOCHROME B(2) - A STUDY WITH WILD-TYPE AND Y143F MUTANT ENZYMES

The crystal structure of flavocytochrome b(2) (L-lactate dehydrogenase ) from Saccharomyces cerevisiae suggests that Tyr143 plays a dual role at the active site: it contributes to substrate binding and, most imp ortantly, makes a hydrogen bond to a heme propionate, which could faci litate communication between the domains. Previous work on the Y143F m utant enzyme provided support for these hypotheses [Miles, C. S., Rouv iere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., Black, M. T ., & Chapman, S. K. (1992) Biochem. J. 285, 187-192; Rouviere-Fourmy, N., Capeillere-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 ob served for the former. A detailed investigation of the phenomenon has shown that, for the wild-type and Y143F forms, lactate at high concent rations inhibits both cytochrome c and ferricyanide reduction. In thes e 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 o f acceptor, flavin and heme reduction are unaffected by high substrate concentrations in the WT enzyme case. For the Y143F mutant, flavin re duction is similarly unaffected, but heme reduction is inhibited to ne arly the same extent by high lactate and acetate concentrations. In th is case, inhibition can probably be ascribed to ionic strength effects . The combination of stopped-flow and steady-state results suggests th at lactate binds with weak affinity at the active site when the flavin is in the semiquinone state, preventing electron transfer to heme b(2 ) and hence to accepters. This phenomenon is analogous to the inhibiti on exerted by pyruvate when bound to the enzyme at the semiquinone sta ge [Tegoni, M., Janot, J. M., & Labeyrie, F. (1990) Eur. J. Biochem. 1 90, 329-342]. We suggest that the substrate carboxylate and the heme p ropionate of the mobile heme-binding domain compete for the Tyr143 hyd roxyl group, hence for approach to the flavin. In the Y143F mutant enz yme, in which the interdomain interaction is impaired, competition wou ld play in favor of the substrate, resulting in the inhibition at lowe r lactate concentrations than observed for the wild-type enzyme.