Studies on the interaction of diphosphopyridine nucleotide analogs with dehydrogenases

Abstract

The addition of the reduced acetylpyridine analog of DPN (APDPNH) to liver alcohol dehydrogenase results in a spectral shift similar to that obtained with DPNH. The spectrum of the reduced pyridine-3-aldehyde analog of DPN (Py-3AlDPNH) is also altered in the presence of the liver enzyme. APDPN and Py-3AlDPN also form hydroxylamine complexes with the liver dehydrogenase. Yeast alcohol dehydrogenase shows no spectral shifts with DPNH or the reduced analogs. DPN and APDPN do not form hydroxylamine complexes with yeast alcohol dehydrogenase. However, Py-3AlDPN forms a hydroxylamine complex with the yeast enzyme which is identical to that observed with the liver alcohol dehydrogenase. Beef heart lactic dehydrogenase produces no detectable shift in the spectrum of DPNH at room temperature; however, under the same conditions an appreciable change in the APDPNH spectrum occurs. The lactic dehydrogenase from skeletal muscle does not influence the spectra of either DPNH, APDPNH, or Py-3AlDPNH. APDPN and charcoal-treated muscle triosephosphate dehydrogenase interact to form a product which is characterized by a maximum at 350 mμ; the spectrum of the APDPN enzyme compound is somewhat more distinct than that obtained with DPN under the same conditions. Iodoacetate abolishes completely the absorption due to DPN, but only will partially remove the extinction with APDPN. The analog also reacts readily with yeast triosephosphate dehydrogenase under circumstances in which DPN appears to be less reactive. Spectral evidence has been obtained that Py-3AlDPN can displace DPN from “native” triosephosphate dehydrogenase of rabbit muscle. Data are presented showing the marked inhibitory action of Py-3AlDPN on the oxidation of triose phosphate and the arsenolysis of acetyl phosphate. The effect of DPN analogs on the arsenolysis reaction appears to be related to the activity of these nucleotides as electron acceptors in promoting the oxidation of triose phosphate. The results of this study are discussed with respect to the nature of the binding of pyridine nucleotides to dehydrogenases.