Blazy B, Thusius D, Baudras A
Biochemistry. 1976 Jan 27;15(2):257-61. doi: 10.1021/bi00647a003.
From a careful steady-state kinetic study it is shown that the inhibition of L-lactate oxidation by cytochrome b2 with ferricyanide as acceptor is of the mixed competitive-noncompetitive type, indicating the formation of an active ternary complex between enzyme, substrate, and inhibitor. With a large excess of acceptor, the simplest formal mechanism consistent with all available data is: E + L equilibrium EL; E + S equilibrium ES leads to EP leads to E + P; ES + L equilibrium ESL leads to EPL leads to EL + P, where L is oxalate, S is L-lactate, P is pyruvate, and E is enzyme. The inhibition kinetics together with the rate constants for oxalate binding to free enzyme (Thusius, D., Blazy, B., and Baudras, A. (1976), Biochemistry, preceding paper in this issue) and recent steady-state experiments on L-lactate deuterated at C-2 (Lederer, F. (1974), Eur. J. Biochem, 46, 393) lead to estimates of some of the elementary rate parameters in the above scheme. As in the case of oxalate (see Thusius et al. reference above), the association rate constant for substrate binding (1.1 x 10(5) M-1 sec-1) is much smaller than a diffusion-controlled value. Our results also imply that dissociation of complex EP to free enzyme and pyruvate is partially rate limiting for the overall reaction.
