The reaction mechanism of the mitochondrial pyridine nucleotide transhydrogenase. A study utilizing arylazido-pyridine nucleotide analogues.
The addition of a purified mitochondrial pyridine nucleotide transhydrogenase enzyme preparation to complex I (NADH-CoQ reductase) results in a significant increase in the NADPH-AcPyAD+ transhydrogenase activity of the complex without influencing the NADH-AcPyAD+ transhydrogenase activity. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of complex I, the purified transhydrogenase enzyme preparation was found to co-migrate with the Mr = 130,000 (130K) subunit of the NADH-CoQ reductase. Loss of the NADPH-NAD+ transhydrogenase activity of complex I following limited tryptic digestion was associated with a corresponding loss of the 130K subunit from the complex. These results suggest that the 130K subunit of complex I is the specific peptide responsible for the catalysis of the NADPH-NAD+ transhydrogenase activity observed in complex I. Studies have been carried out testing the influence of photoaffinity pyridine nucleotide probes on the NADPH-NAD+ transhydrogenase activity catalyzed at three levels of resolution, i.e. a homogeneous transhydrogenase preparation, a partially resolved membrane preparation (complex I), and an intact mitochondrial membrane preparation (EDTA particles). Such studies have revealed arylazido-beta-alanyl NADP+ (N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+) to be a potent inhibitor and an active site-directed reagent for NADPH-NAD+ transhydrogenation at all three levels of resolution. On the other hand, arylazido-beta-alanyl NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)-amino]propionyl)NAD+ does not produce a significant degree of inhibition of NADPH-NAD+ transhydrogenase activities prior to or following photoirradiation. Nevertheless, the NAD+ analogue has been found to specifically label, covalently, the transhydrogenase protein following photoirradiation of an enzyme-analogue mixture. Arylazido-beta-alanyl NAD+ can as well function as a substrate during transhydrogenation by virtue of being able to accept a hydride ion from NADPH. An interpretation of the observed nucleotide photoprobe specificity for interaction at the active site for transhydrogenation is advanced. In this interpretation, an ordered binding of substrate involves an initial NADP(H) (or NADP+ photoprobe) interaction with a hydrophobic region at the transhydrogenation site. This initial reactivity is followed by a positioning of NAD(H) (or the NAD+ photoprobe analogue) above or periphery to the NADP(H) nucleotide present at the active site region. Supportive evidence for this model for transhydrogenation is presented and discussed.
D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D001665:Binding Sites; D002417:Cattle; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD / Q000031:analogs & derivatives* / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives* / Q000494:pharmacology; D009250:NADP Transhydrogenases / Q000378:metabolism*; D011485:Protein Binding; D013367:Submitochondrial Particles / Q000201:enzymology*; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; C020606:arylazido-beta-alanyl-NAD; D009243:NAD; D009249:NADP; C023549:arylazido-beta-alanyl-NADP; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases; D014357:Trypsin