title: Binding studies of a spin-labelled oxidized coenzyme to bovine-liver glutamate dehydrogenase.
abstract: NAD+ with a nitroxide piperidine ring linked to the NH2 group of the adenine possesses full coenzymatic activity with glutamate dehydrogenase. Electron spin resonance spectra in the presence of glutamate dehydrogenase show mixtures of free and strongly immobilized spin-label. Binding studies in phosphate buffer demonstrate: (a) weak binary binding to the enzyme with a dissociation constant in the order of 2mM;(b) an indication for negative cooperativity or different sites for binding to enzyme-2-oxoglutarate, with dissociation constants in the order of 20--250muM; (c) similar but much weaker binding to enzyme-2-oxoglutarate-ADP; (d) a strong positive cooperative binding to enzyme-2-oxoglutarate-GTP, dependent on the enzyme concentration. Binding of phosphate to the enzyme with a Kd of about 20 mM or binding of pyrophosphate or tripolyphosphate with a Dd of about 2.5 mM enhances the binding of spin-labelled NAD+ in the presence of 2-oxoglutarate. There is evidence that the binding sites for these phosphates coincide with phosphate binding subsites of GTP.
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D011756:Diphosphates / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D007656:Ketoglutaric Acids; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD; D010710:Phosphates; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels
chemical_list: D011756:Diphosphates; D007656:Ketoglutaric Acids; D010710:Phosphates; D013113:Spin Labels; D009243:NAD; D005969:Glutamate Dehydrogenase