Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893

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Abstract
The mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer Mr 110 000. It is located in the inner mitcohondrial membrane and catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner membrane. THe amino acid sequence and the nucleotide binding sites of the enzyme have been determined [Yamaguchi M., Hatefi, Y., Trach, K., and Hoch, J. A. (1988) J. Biol. Chem. 263, 2761; Wakabayashi, S., and Hatefi, Y. (1987) Biochem. Int. 15, 915-924]. N-Ethylmaleimide, as well as other sulfhydryl group modifiers, inhibits the transhydrogenase. The presence of NADP in the incubation mixture suppressed the inhibition rate by N-ethylmaleimide, and the presence of NADPH greatly increased it. NAD and NADH had little or no effect. The NADPH effect was concentration dependent and saturable, with a half-maximal NADPH concentration effect close to the Km of the enzyme for NADPH. Study of the effect of pH on the N-ethylmaleimide inhibition rate showed that NADPH binding by the enzyme lowers the apparent pKa of the N-ethylmaleimide-sensitive group by 0.4 of a pH unit and NADP binding raises this pKa by 0.4 of a pH unit, thus providing a rational for the effects of NADP and NADPH on the N-ethylmalemide inhibition rate. With the use of N-[3H]ethylmaleimide, the modified sulfhydryl group involved in the NADP(H)-modulated inhibition of the transhydrogenase was identified as that belonging to Cys-893, which is located 113 residues upstream of the tryosyl residue modified by [p-(fluorosulfonyl)benzoyl]-5''-adenosine at the putative NADP(H) binding site of the enzyme (see above references). In addition adata have been presented suggesting that Cys-893 is not an essential residue and that enzyme inhibition is partial when the modifier is methyl methanethiosulfonate which introduces a smaller group (-SCH3) at the modified cysteine sulfhydryl than does N-ethylmaleimide. However, regardless of its role in catalysis, Cys-893 has provided a means to show that substrate and product binding cause opposite conformation changes in the transhydrogenase. This is the first instance that such a phenomenon, which has important mechanistic implications regarding proton pumping, has been demonstrated for any energy-transducing enzyme of the mitochondrial or bacterial oxidative phosphorylation system.