Effect of sulfhydryl modifications on transhydrogenase

The effect of various sulfhydryl modification agents on the NADPH..-->..AcPyAD activity of bovine heart pyridine dinucleotide transhydrogenase in the presence and absence of substrates was investigated. Copper (o-phenanthroline)/sub 2/, (Cu(OP)/sub 2/); 5,5'-dithiobis-(2-nitrobenzoic acid), (DTNB); and N-ethylmaleimide (NEM) were able to completely inhibit the activity of transhydrogenase in the absence of substrates. Inhibition of DTNB and Cu(OP)/sub 2/ was accompanied by a change in mobility on SDS-PAGE of approximately 2000 Daltons. NADP/sup +/ provided protection against Cu(OP)/sub 2/, DTNB and NEM while NADPH protected against Cu(OP)/sub 2/ and DTNB. NAD(H) provided no protection against inactivation. The effects of DTNB and Cu(OP)/sub 2/ were reversible upon addition of dithiothreitol and BETA-mercaptoethanol. It was previously shown by DTNB titration that there were two sulfhydryls exposed on the surface and four buried in the native enzyme. The use of Cu(OP)/sub 2/, DTNB and NEM indicate that one sulfhydryl is contained within the NADP-binding site and the other is peripheral to this site, but not in the NAD-binding site, and that these two sulfhydryls are spatially vicinal.