Interaction of purified nicotinamide nucleotide transhydrogenase with dicyclohexylcarbodiimide

Abstract

The inhibition of the energy-linked nicotinamidenucleotide transhydrogenase (TH; EC 1.6.1.1) by dicyclohexylcarbodiimide (DCCD) was further studied because of its important mechanistic implications. It was shown earlier that TH bound to submitochondrial particles from bovine heart is inhibited by DCCD and that NAD(H) protects the enzyme against this inhibition. Pennington and Fisher working with purified TH concluded that NAD(H) does not protect against DCCD inhibition and that DCCD inhibition involves the TH proton channel rather than the nucleotide-binding active site. The present study shows that NAD(H) as well as AMP and ADP, which are known to bind to the NAD(H) binding site from competitive inhibition studies, protect the purified TH against inhibition by DCCD, whereas 2''-AMP and 3''-AMP, which bind to the NADP(H) site on TH, do not protect. In addition, it is shown that whereas the unmodified TH binds to NAD-agarose such that it is elutable by buffer containing NADH, the DCCD-modified enzyme does not bind to NAD-agarose. These results suggest strongly that DCCD binds at or near the NAD(H) binding site on TH. Another less likely possibility is that NAD(H) and DCCD bind to separate sites, but their bindings are mutually exclusive. With the use of [14C]DCCD, it has been shown that 100% activity inhibition corresponds to 0.5 mol of DCCD binding/mol of TH (MW .apprx. 11 .times. 104). Both the inhibition and DCCD binding are pseudo 1st order with respect to the time of TH exposure to DCCD and follow a parallel course when binding is plotted on the basis of saturation at 0.5 mol of DCCD/mol of TH. Since the purified TH and the membrane-bound TH appear to be dimeric, these results suggest a case of half-of-the-sites reactivity in which only the active protomer is capable of rapid DCCD binding. This conclusion agrees with the possibility of DCCD and NAD(H) binding at the same site. Thus, the protomer that cannot bind DCCD might be inactive because it cannot bind NAD(H) either.