Ghobadi S, Nemat-Gorgani M, Golabi S M, Zare H R, Moosavi-Movahedi A A
Institute of Biochemistry and Biophysics, University of Tehran, Iran.
J Enzyme Inhib. 2000;15(5):497-508. doi: 10.3109/14756360009040705.
The effects of nickel ions on reductive amination and oxidative deamination activities of bovine liver glutamate dehydrogenase (GDH) were examined kinetically by UV spectroscopy, at 27 degrees C, using 50 mM Tris, pH 7.8, containing 0.1 M NaCl. Kinetic analysis of the data obtained by varying NADH concentration indicated strong inhibition, presumably due to binding of the coenzyme to the regulatory site. In contrast, almost no inhibition was observed in the forward reaction. The fact that nickel ions have the capacity to enhance binding of NADH to the enzyme was confirmed by an electrochemical method using a modified glassy carbon electrode. Use of NADPH instead of NADH showed only a weak substrate inhibition, presumably related to lower affinity of NADPH for binding to the regulatory site. Lineweaver-Burk plots with respect to alpha-ketoglutarate and ammonium ions indicated substrate and competitive inhibition patterns in the presence of nickel ions, respectively. ADP at 0.2 mM concentration protected inhibition caused by nickel. These observations are explained in terms of formation of a nickel-NADH complex with a higher affinity for binding to the regulatory site in GDH, as compared with the situation where nickel is not present. Such effects may be important for regulation of GDH and other NADH-utilizing enzymes.
在27℃下，使用含有0.1M NaCl的50mM Tris（pH 7.8），通过紫外光谱法对镍离子对牛肝谷氨酸脱氢酶（GDH）的还原胺化和氧化脱氨活性的影响进行了动力学研究。通过改变NADH浓度获得的数据的动力学分析表明存在强烈抑制作用，推测这是由于辅酶与调节位点结合所致。相比之下，在前向反应中几乎未观察到抑制作用。使用修饰的玻碳电极通过电化学方法证实了镍离子具有增强NADH与该酶结合的能力。使用NADPH代替NADH仅显示出较弱的底物抑制作用，推测这与NADPH与调节位点结合的亲和力较低有关。关于α-酮戊二酸和铵离子的Lineweaver-Burk图分别表明在镍离子存在下底物抑制和竞争性抑制模式。0.2mM浓度的ADP可保护免受镍引起的抑制作用。这些观察结果可以解释为与不存在镍的情况相比，形成了对GDH调节位点具有更高结合亲和力的镍-NADH复合物。这种效应可能对GDH和其他利用NADH的酶的调节很重要。
