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 [EC 1.4.1.3]. ESR spectra in the presence of glutamate dehydrogenase show mixtures of free and strongly immobilized spin-label. Binding studies in phosphate buffer demonstrate weak binary binding to the enzyme with a Kd in the order of 2 mM, an indication for negative cooperativity or different sites for binding to enzyme .cntdot. 2-oxoglutarate, with Kd in the order of 20-250 .mu.M similar but much weaker binding to enzyme.cntdot.2-oxoglutarate.cntdot.ADP a strong positive cooperative binding to enzyme.cntdot.2-oxoglutarate.cntdot.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 Kd of about 2.5 mM enhances the binding of spin-labeled NAD+ in the presence of 2-oxoglutarate. There is evidence that the binding sites for these phosphates coincide with phosphate binding subsites of GTP.