A shift in the equilibrium constant at the catalytic site of proton-translocating transhydrogenase: significance for a 'binding-change' mechanism

In mitochondria and bacteria, transhydrogenase uses the transmembrane proton gradient (Delta p) to drive reduction of NADP(+) by NADH. We have investigated the pre-steady-state kinetics of NADP(+) reduction by acetylpyridine adenine dinucleotide (AcPdADH, an analogue of NADH) in complexes formed from. the two, separately prepared, recombinant, peripheral subunits of the enzyme: the dI component, which binds NAD(+) and NADH, and the dIII component, which binds NADP(+) and NADPH. In the stopped-flow spectrophotometer the reaction proceeds as a single-turnover burst of hydride transfer to NADP(+) on dIII before product NADPH release becomes limiting in steady state. The burst is biphasic. The results indicate that the fast phase represents direct hydride transfer from AcPdADH to NADP(+) in dI:dIII complexes, and that the slow phase, which predominates when [dI] < [dIII], corresponds to dissociation of the protein complexes during multiple turnovers of dI. Measurements on the amplitude of the burst, and on the apparent first-order rate constant of the fast phase, indicate that the equilibrium constant of the hydride-transfer step on the enzyme is shifted relative to that in solution. This has consequences for a model proposed earlier, in which dp is used, not at the hydride-transfer step, but to change the binding affinities of NADP(+) and NADPH.