Conformational features of bovine heart mitochondrial transhydrogenase

Abstract

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)

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

“…As pointed out above, treatment of the bovine mitochondrial nicotinamide nucleotide transhydrogenase with NADPH increases the sensitivity of the enzyme to inhibition by a number of reagents, especially NEM and trypsin (Blazyk et al, 1976; Earle et al, 1978). These findings have been interpreted to mean that NADPH binding brings about a conformational change of the enzyme, which results in greater accessibility of the target moieties or bonds to modification by the added reagents (Blazyk et al, 1976;Earle et al, 1978;Modrak et al, 1988). The studies reported in this paper have shown that in the presence of NADPH a single sulfhydryl group belonging to cysteine-893 becomes highly susceptible to modification by NEM.…”

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase: effect of substrates on the sensitivity of the enzyme to trypsin and identification of tryptic cleavage sites

“…The stimulatory effect of NADPH is not confined to trypsin sensitivity of the enzyme. It has also been reported in connection with inhibition of the transhydrogenase by thiol modifiers (Earle et al, 1978;Modrak et al, 1988;Yamaguchi & Hatefi, 1989), dicyclohexylcarbodiimide (Phelps & Hatefi, 1984), and ethoxyformic anhydride and dansyl chloride (Yamaguchi & Hatefi, 1985). Our recent studies with /V-ethylmaleimide as the inhibitor have shown that NADPH accelerates and NADP retards the inhibition rate, and that the pKt of the target residue (Cys-893) changes from 9.1 in the absence of added substrates to 8.7 in the presence of NADPH and to 9.5 in the presence of NADP (Yamaguchi & Hatefi, 1989).…”