Mitochondrial nicotinamide nucleotide transhydrogenase: nonidentical modification by N,N'-dicyclohexylcarbodiimide and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline at the NAD(H) binding site.
The energy-linked nicotinamide nucleotide transhydrogenase (TH) purified from bovine heart mitochondria is inhibited by the carboxyl group modifiers, N,N'-dicyclohexylcarbodiimide (DCCD) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ). With either reagent, complete activity inhibition corresponds to modification of one carboxyl group per 2 mol (monomers) of this dimeric enzyme, suggesting half-site reactivity toward DCCD and EEDQ [D. C. Phelps, and Y. Hatefi (1984) Biochemistry 23, 4475-4480; 6340-6344]. It has also been shown in the former reference that DCCD appears to modify TH at the NAD(H)-binding site. The present paper presents data suggesting that EEDQ also binds at or near the NAD(H)-binding domain of TH, but at a site not identical to that of DCCD: TH modified with and inhibited approximately 85% by EEDQ could be further labeled with [14C]DCCD to the extent of 70% of the maximum in the same time period that unmodified TH was modified by [14C]DCCD to near saturation (1 mol DCCD/TH dimer); DCCD-modified TH did not bind to NAD-agarose, while EEDQ-modified TH showed partial affinity for NAD-agarose; 5'-AMP completely protected TH against modification by DCCD, but showed only a weak protective effect against EEDQ; by contrast, NMNH, which is a TH substrate and binds to the NADH site, did not protect TH against DCCD, but completely protected the enzyme against attack by EEDQ. The results are consistent with the possibility that DCCD modifies TH where the 5'-AMP moiety of NAD(H) binds, while EEDQ modifies the enzyme where the NMN(H) moiety of NAD(H) resides.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002234:Carbodiimides / Q000494:pharmacology*; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000378:metabolism*; D011804:Quinolines / Q000494:pharmacology*; D012685:Sepharose
D002234:Carbodiimides; D011804:Quinolines; D009243:NAD; D000249:Adenosine Monophosphate; D004024:Dicyclohexylcarbodiimide; C000470:EEDQ; D012685:Sepharose; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases