Mitochondrial nicotinamide nucleotide transhydrogenase: inhibition by ethoxyformic anhydride, dansyl chloride, and pyridoxal phosphate.
Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH; EC 1.6.1.1) was inactivated by treatment with pyridoxal phosphate, ethoxyformic anhydride (EFA) or dansyl chloride. NADP and NADPH, but not NAD and NADH, protected TH against inhibition by pyridoxal phosphate, and L-lysine reversed this inhibition. The results suggested modification of an essential lysyl residue by pyridoxal phosphate, possibly at the NADP(H) binding site of TH. EFA and dansyl chloride inhibited TH in a similar manner. The effect of pH on the rate of inhibition of TH by EFA and dansyl chloride was the same, and in both cases addition of NADP and particularly NADPH accelerated the rate of inhibition, while addition of NAD or NADH had no effect. Double inhibition studies, using in one experiment dithiothreitol-reversible inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) to protect the thiol groups of TH, and in another experiment lysine-reversible inhibition by pyridoxal phosphate to protect the putative essential lysyl residues of the enzyme, followed in each case by further treatment of the protected TH with EFA or dansyl chloride, suggested that the inhibitions by EFA and dansyl chloride were independent of the inhibitions by 5,5'-dithiobis (2-nitrobenzoic acid) and pyridoxal phosphate. The inhibitors discussed above are interesting, because pyridoxal phosphate is the only reagent known which appears to modify an essential residue in the NADP(H), but not the NAD(H), binding site of TH, and EFA and dansyl chloride are the only inhibitors known which appear to react with essential residues outside the active site of TH. It is possible that EFA and dansyl chloride inhibitions involve modification of essential prototropic residues in the proton translocation domain of the enzyme.
D000818:Animals; D003619:Dansyl Compounds / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005561:Formates / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors*; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000037:antagonists & inhibitors*; D011732:Pyridoxal Phosphate / Q000494:pharmacology*
D003619:Dansyl Compounds; D005561:Formates; D009243:NAD; D009249:NADP; D004024:Dicyclohexylcarbodiimide; D011732:Pyridoxal Phosphate; D004228:Dithionitrobenzoic Acid; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases; D004047:Diethyl Pyrocarbonate; C029038:dansyl chloride