Saha S K, Maniscalco S J, Fisher H F
Laboratory of Molecular Biochemistry, Veterans Affairs Medical Center, Kansas City, Missouri, USA.
Biochemistry. 1996 Dec 24;35(51):16483-8. doi: 10.1021/bi9612531.
We have related the ratios of the protein fluorescence quenching and nucleotide absorbance time courses for the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate to identify the occurrence and sequential location of a previously demonstrated charge-transfer intermediate. Static studies showed the major portion of the fluorescence quenching signal to be due to radiationless singlet energy transfer from tryptophan to reduced coenzyme chromophores and that conformational changes contribute little to this signal. The ratio approach applied to the transient time courses shows correspondingly that, over most of the time range, the fluorescence quenching signal provides a quantitative measure of the sum of all posthydride transfer species. However, it also indicates the very early occurrence of a species of anomalous optical properties for the reaction catalyzed by the Clostridium symbiosum enzyme as well as that from bovine liver. Transient-state kinetic isotope effect time courses of both the fluorescence and the absorbance signals confirm that this species must be the prehydride charge-transfer complex in both enzyme reactions. Kinetic analysis of alpha-deuterio- and alpha-protio-L-glutamate reaction time courses proves the kinetic competence of the assignments. These results also demonstrate that the intramolecular transfer of a proton from the alpha-amino group of the substrate to an immediately adjacent aspartate carboxylate group on the enzyme is an obligatory initial event in the reactions catalyzed by both enzyme species, even though the occurrence of protein release from a critical lysine residue to the solvent occurs at different phases in those two reactions. The abnormally low intrinsic KIE required to simulate both the alpha-deuterio-L-glutamate reaction and its protio counterpart implies that the transition state of the hydride transfer step must be highly asymmetric.
我们已将谷氨酸脱氢酶催化L-谷氨酸氧化脱氨反应中蛋白质荧光猝灭与核苷酸吸光度时间进程的比率关联起来，以确定先前已证实的电荷转移中间体的出现及其顺序位置。静态研究表明，荧光猝灭信号的主要部分是由于色氨酸到还原辅酶发色团的无辐射单线态能量转移，且构象变化对该信号的贡献很小。应用于瞬态时间进程的比率方法相应地表明，在大部分时间范围内，荧光猝灭信号提供了所有氢化物转移后物种总和的定量测量。然而，它也表明，对于共生梭菌酶以及牛肝酶催化的反应，存在一种具有异常光学性质的物种，且该物种出现得非常早。荧光和吸光度信号的瞬态动力学同位素效应时间进程证实，在这两种酶反应中，该物种必定是氢化物转移前的电荷转移复合物。对α-氘代和α-质子化L-谷氨酸反应时间进程的动力学分析证明了这些归属的动力学可行性。这些结果还表明，在这两种酶催化的反应中，底物α-氨基上的质子向酶上紧邻的天冬氨酸羧基的分子内转移是一个必需的初始事件，尽管在这两个反应中，关键赖氨酸残基向溶剂释放蛋白质的过程发生在不同阶段。模拟α-氘代-L-谷氨酸反应及其质子化对应反应所需的异常低的固有动力学同位素效应意味着氢化物转移步骤的过渡态必定是高度不对称的。
