Tally Jon F, Maniscalco Steven J, Saha Swapan K, Fisher Harvey F
Laboratory of Molecular Biochemistry, Veteran Affairs Medical Center, and Department of Biochemistry, University of Kansas Medical Center, Kansas City, Missouri 64128, USA.
Biochemistry. 2002 Sep 17;41(37):11284-93. doi: 10.1021/bi0202068.
We present a multiwavelength, transient-state kinetic study of the oxidative deamination reaction catalyzed by Clostridium symbiosum glutamate dehydrogenase (csGDH) producing the real-time reaction courses of spectroscopically resolved kinetically competent intermediate complexes. The results show striking differences from a corresponding transient-state study of the same reaction by the structurally homologous enzyme from beef liver (blGDH). In addition to the highly blue-shifted alpha-iminoglutarate and highly red-shifted carbinolamine complexes observed in both reactions, the csGDH reaction appeared to show the release of free NADH at a very early and mechanistically unlikely point in the reaction. Using lactic acid dehydrogenase as a "reporter" for free NADH, we show that the early portion of this signal reflects previously unobserved spectrally unshifted enzyme-bound NADH complexes. We provide experimental evidence to show that such spectrally anomalous complexes must represent forms of the known alpha-imino and alpha-carbinolamine complexes in which the active site cleft is open. This evidence includes isothermal calorimetric measurements and pH-jump experiments that show the existence of differing two-state transitions in blGDH and csGDH and locate active site domain motions at differing points in the transient-state time courses of the two enzyme reactions. We prove the kinetic competence of a new and more highly detailed mechanism for the csGDH reaction that involves the alternation of open and closed enzyme complexes as integral steps. These findings, supported by the available X-ray crystal structure data, suggest the existence of a programmed time course of protein domain motions coordinated with the classically considered chemical time course. This new viewpoint may be presumed to be applicable to enzyme reactions other than those of the alpha-amino acid dehydrogenases.
我们对共生梭菌谷氨酸脱氢酶（csGDH）催化的氧化脱氨反应进行了多波长瞬态动力学研究，得出了光谱分辨的动力学活性中间复合物的实时反应过程。结果显示，该反应与来自牛肝的结构同源酶（blGDH）对同一反应进行的相应瞬态研究存在显著差异。除了在两个反应中均观察到的高度蓝移的α-亚氨基戊二酸和高度红移的甲醇胺复合物外，csGDH反应似乎在反应的非常早期且从机理上不太可能的点就显示出游离NADH的释放。使用乳酸脱氢酶作为游离NADH的“报告分子”，我们表明该信号的早期部分反映了以前未观察到的光谱未发生位移的酶结合NADH复合物。我们提供实验证据表明，这种光谱异常的复合物必定代表已知的α-亚氨基和α-甲醇胺复合物的形式，其中活性位点裂隙是开放的。该证据包括等温量热测量和pH跃变实验，这些实验表明blGDH和csGDH中存在不同的双态转变，并在两种酶反应的瞬态时间过程中的不同点定位了活性位点结构域的运动。我们证明了一种新的、更详细的csGDH反应机制的动力学活性，该机制涉及开放和封闭酶复合物的交替作为整体步骤。这些发现得到了现有的X射线晶体结构数据的支持，表明存在与经典化学时间过程相协调的蛋白质结构域运动的程序性时间过程。可以推测，这种新观点可能适用于除α-氨基酸脱氢酶以外的酶反应。
