Dicyclohexylcarbodiimide modification of bovine heart mitochondrial transhydrogenase.
Dicyclohexylcarbodiimide (DCCD) inhibits the reduction of oxidized 3-acetylpyridine adenine dinucleotide (AcPyAD+) by NADPH catalyzed by purified and bovine heart submitochondrial particle transhydrogenase. Kinetic studies demonstrate that the modification of 1 residue results in complete inactivation. Both transhydrogenase preparations were labeled with [14C]DCCD. Labeling of the purified enzyme was time-dependent and paralleled the extent of inhibition. The incorporation of approximately 1 mol of [14C]DCCD/monomer resulted in complete inactivation of the enzyme. At longer preincubation times or at higher DCCD concentrations, more than 1 mol of DCCD reacted and cross-linked dimers of transhydrogenase were formed. The effect of substrates on DCCD inactivation was investigated. AcPyAD+ provided no protection, and NADH gave partial protection in submitochondrial particles, but not of the purified enzyme. NADPH and NADP+ stimulated inhibition. These results indicate that DCCD modification occurs outside the active site. In experiments with transhydrogenase reconstituted into K+-loaded phosphatidylcholine liposomes, DCCD inhibited the rate of H+ uptake into the vesicles to a significantly greater extent than transhydrogenation. The incorporation of approximately 1 mol of DCCD/mol of transhydrogenase monomer completely inhibited H+ translocation, whereas complete inactivation of hydride ion transfer accompanied the incorporation of approximately 2 mol of DCCD. These results indicate that DCCD modified transhydrogenase outside the active site, possibly in a putative H+-binding domain that functions to translocate protons across the membrane by a pump rather than by a loop mechanism.
D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000378:metabolism*; D011485:Protein Binding; D013367:Submitochondrial Particles / Q000201:enzymology*
D002234:Carbodiimides; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D004024:Dicyclohexylcarbodiimide; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases