Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893.
The mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer Mr 110,000. It is located in the inner mitochondrial 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., & Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767; Wakabayashi, S., & 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 rationale for the effects of NADP and NADPH on the N-ethylmaleimide 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 tyrosyl residue modified by [p-(fluorosulfonyl)benzoyl]-5'-adenosine at the putative NADP(H) binding site of the enzyme (see above references).(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Basic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, California 92037.
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Yamaguchi
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Hatefi
D000818:Animals; D002417:Cattle; D003545:Cysteine; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP; D009250:NADP Transhydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity
D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases; D003545:Cysteine; D005033:Ethylmaleimide