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)
Yamaguchi
M
M
Department of Basic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, California 92037.
Hatefi
Y
Y
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