Zantema A, Trommer W E, Wenzel H, Robillard G T
Eur J Biochem. 1977 Jan 3;72(1):175-84. doi: 10.1111/j.1432-1033.1977.tb11237.x.
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.
与连接到腺嘌呤氨基上的氮氧哌啶环相连的NAD⁺对谷氨酸脱氢酶具有完全的辅酶活性。在谷氨酸脱氢酶存在下的电子自旋共振光谱显示出自由和强固定化自旋标记的混合物。在磷酸盐缓冲液中的结合研究表明：（a）与酶的弱二元结合，解离常数约为2mM；（b）表明与酶 - 2 - 氧代戊二酸结合存在负协同性或不同位点，解离常数约为20 - 250μM；（c）与酶 - 2 - 氧代戊二酸 - ADP的结合相似但弱得多；（d）与酶 - 2 - 氧代戊二酸 - GTP的强正协同结合，取决于酶浓度。磷酸盐以约20mM的Kd与酶结合，或焦磷酸盐或三聚磷酸盐以约2.5mM的Dd与酶结合，在2 - 氧代戊二酸存在下增强了自旋标记的NAD⁺的结合。有证据表明这些磷酸盐的结合位点与GTP的磷酸盐结合亚位点重合。
