31 P nuclear magnetic resonance studies of the interaction of pyridine nucleotide coenzymes with dehydrogenases.
31P nuclear magnetic resonance spectra of the pyrophosphate group in NAD+ and NADH were recorded in the presence of beef heart lactate dehydrogenase and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. At high lactate dehydrogenase concentrations (60 mg/ml), two NADH resonances are observed: a slowly exchanging peak which is shifted to 1.9 ppm downfield (relative to free NADH) and a rapidly exchanging peak with a downfield shift of 0.5-0.6 ppm. At lover concentrations (15 mg/ml) only the rapidly exchanging peak is observed thus indicating that the peak observed at-1.9 ppm is due to coenzyme bound to an aggregated enzyme species. With NAD+, rapid exchange and downfield shifts are observed at both enzyme and concentrations, with shifts of about 1.5 ppm and 0.6 ppm at 60 and 15 mg/ml, respectively. In the presence of glyceraldehydephosphate dehydrogenase, the results are independent of enzyme concentration, and slow exchange and upfield shifts of 0.4-0.6 ppm occur with each coenzyme. These data indicate that the environment of the pyrophosphate group of oxidized and reduced coenzyme is the same for a given dehydrogenase, but is different in one enzyme from the other. The resonances observed with glyceraldehydephosphate dehydrogenase are broader than those observed with lactate dehydrogenase. This is indicative of either shorter relaxation times with the former enzyme, or the presence of multiple, unresolved resonances.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009243:NAD; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits
D009243:NAD; D007770:L-Lactate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases