Studies on sulfhydryl group modification of mitochondrial pyridine dinucleotide transhydrogenase.

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)

Section: Discussionsupporting

confidence: 83%

mentioning

confidence: 99%

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

Yamaguchi¹,

Hatefi²

1989

“…The pKa for the reactive sulfhydryl group of the purified enzyme was determined to be ~8.6. This differs markedly from the previously reported pKi for SMP transhydrogenase of 7.4 (O'Neal & Fisher, 1977). The enzyme as purified in this work is stable up to pH 8.6 with less than 10% of the initial activity lost in 15 min.…”

Section: Dtnb Modification Of Purified and Reconstitutedcontrasting

confidence: 99%

“…These results indicate that DTNB does not react at the NAD-binding site since neither NAD+ nor NADH provided protection and only NADP+ is effective in protecting against DTNB inactivation. In contrast, O'Neal and Fisher (1977) found both NADP+ and NADPH to protect the enzyme from DTNB inactivation in SMP. Figure 2 shows the pH depenency for DTNB inactivation (open circles).…”

Section: Dtnb Modification Of Purified and Reconstitutedmentioning

confidence: 72%

Conformational features of bovine heart mitochondrial transhydrogenase

Modrak¹,

Wu²,

Alberta³

et al. 1988

Both purified and functionally reconstituted bovine heart mitochondrial transhydrogenase were treated with various sulfhydryl modification reagents in the presence of substrates. In all cases, NAD+ and NADH had no effect on the rate of inactivation. NADP+ protected transhydrogenase from inactivation by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in both systems, while NADPH slightly protected the reconstituted enzyme but stimulated inactivation in the purified enzyme. The rate of N-ethylmaleimide (NEM) inactivation was enhanced by NADPH in both systems. The copper-(o-phenanthroline)2 complex [Cu(OP)2] inhibited the purified enzyme, and this inhibition was substantially prevented by NADP+. Transhydrogenase was shown to undergo conformational changes upon binding of NADP+ or NADPH. Sulfhydryl quantitation with DTNB indicated the presence of two sulfhydryl groups exposed to the external medium in the native conformation of the soluble purified enzyme or after reconstitution into phosphatidylcholine liposomes. In the presence of NADP+, one sulfhydryl group was quantitated in the nondenatured soluble enzyme, while none was found in the reconstituted enzyme, suggesting that the reactive sulfhydryl groups were less accessible in the NADP+-enzyme complex. In the presence of NADPH, however, four sulfhydryl groups were found to be exposed to DTNB in both the soluble and reconstituted enzymes. NEM selectively reacted with only one sulfhydryl group of the purified enzyme in the absence of substrates, but the presence of NADPH stimulated the NEM-dependent inactivation of the enzyme and resulted in the modification of three additional sulfhydryl groups. The sulfhydryl group not modified by NEM in the absence of substrates is not sterically hindered in the native enzyme as it can still be quantitated by DTNB or modified by iodoacetamide.(ABSTRACT TRUNCATED AT 250 WORDS)

“…N-Ethylmaleimide has been employed to localize sulfhydryl groups in both the NADand NADP-binding sites of Escherichia coli transhydrogenase (Houghton et al, 1976). However, the active-site sulfhydryl group in bovine heart transhydrogenase (O'Neal & Fisher, 1977) is not susceptible to MalNEt modification (Earle et al, 1978b). Inactivation with p-chloromercuribenzoate demonstrated the presence of an essential sulfhydryl group on the R. rubrum transhydrogenase soluble factor (Koenings & Guillory, 1973).…”

Section: Resultsmentioning

confidence: 99%

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase: chemical modification with N-ethylmaleimide and 2,4-pentanedione

Jacobs¹,

Fisher²

1979

Rhodospirillum rubrum chromatophore pyridine dinucleotide transhydrogenase complex, which catalyzes the reversible transfer of a hydride ion equivalent between the 4A locus of NAD and the 4B locus of NADP, consists of an integral membrane-bound component and a soluble peripheral protein factor. N-Ethylmaleimide inactivated both separated transhydrogenase components. A NADP-binding site on both components [McFadden, B. J., & Fisher, R. R. (1978) Arch. Biochem. Biophys. 190, 820-8281 is confirmed by NADP' protection against inactivation. NADPH and NAD' did not affect N-ethylmaleimide modification of either component. Modification of the intact transhydrogenase complex was promoted by NADPH, partially prevented by NADP', and unaffected by NAD'. NADPH selectively enhanced the modification of the complexed soluble factor. 2,CPentanedione inactivation of the transhydrogenase complex and membrane component was partially prevented by NADP' or NADPH but not affected by NAD'. Substrates did not influence the inactivation of the soluble factor. Hydroxylamine treatment reversed inactivation of the separated components, suggesting that inactivation results from modification of lysyl residues.