Saha S K, Maniscalco S J, Singh N, Fisher H F
Department of Biochemistry, University of Kansas Medical Center, Kansas City.
J Biol Chem. 1994 Nov 25;269(47):29592-7.
In previous transient state kinetic work from this laboratory, we proposed a new mechanism for the glutamate dehydrogenase-catalyzed oxidative deamination reaction involving an initial replacement of a proton from lysine 126 by a single bound water molecule, followed by closure of the active site cleft and expulsion of bulk water, providing a hydrophobic environment for the ensuing hydride transfer step. Here, we report the results of further transient state fluorescence, absorbance, and kinetic isotope effect studies, which demonstrate the occurrence of an unusual intermediate in the early steps of that reaction. This phenomenon is revealed by an initial fluorescence burst that occurs in the time period where the absorbance signal is still in its lag phase. Using an extension of the proton/product ratio approach we have described earlier, we show that this intermediate is a strongly fluorescent but weakly absorbing species whose absorption maximum is red-shifted beyond that of other known complexes of this enzyme. The transient state kinetic isotope effects of the fluorescence and absorbance signals are compatible only with a reaction scheme in which the formation of the fluorescent complex precedes the hydride transfer step. The optical properties of this enzyme-oxidized coenzyme-substrate intermediate strongly suggest that it is a charge-transfer complex, similar in nature to the complex responsible for the well known "Racker band" reported in 1952 for glyceraldehyde-3-phosphatase dehydrogenase (Racker, E., and Krimsky, I. (1952) Nature 169, 1043-1044). The crystal structure studies of the enzyme-coenzyme and enzyme-L-glutamate complexes of the closely analogous Clostridium symbosium glutamate dehydrogenase, reported by the Sheffield group (Stillman, T. J., Baker, P. J., Britton, K. L., and Rice, D. W. (1993) J. Mol. Biol. 234, 1131-1139), provide a basis for a physical explanation of the phenomenon. We conclude that the charge transfer phenomenon is caused by the near apposition of the unprotonated alpha-amino group of the substrate in a form of the enzyme in which a conformational change has caused the complete closing of the active site cleft.
在本实验室之前关于瞬态动力学的研究中，我们提出了一种谷氨酸脱氢酶催化的氧化脱氨反应的新机制，该机制涉及赖氨酸126上的一个质子首先被单个结合水分子取代，随后活性位点裂隙关闭并排出大量水，为随后的氢化物转移步骤提供疏水环境。在此，我们报告了进一步的瞬态荧光、吸光度和动力学同位素效应研究结果，这些研究表明在该反应的早期步骤中出现了一种异常中间体。这种现象通过在吸光度信号仍处于滞后阶段的时间段内出现的初始荧光猝发来揭示。使用我们之前描述的质子/产物比方法的扩展，我们表明这种中间体是一种荧光强但吸收弱的物种，其最大吸收峰发生红移，超过了该酶其他已知复合物的最大吸收峰。荧光和吸光度信号的瞬态动力学同位素效应仅与一种反应方案兼容，即在该方案中荧光复合物的形成先于氢化物转移步骤。这种酶 - 氧化辅酶 - 底物中间体的光学性质强烈表明它是一种电荷转移复合物，其性质类似于1952年报道的甘油醛 - 3 - 磷酸脱氢酶（Racker, E., and Krimsky, I. (1952) Nature 169, 1043 - 1044）中著名的“Racker带”所对应的复合物。谢菲尔德小组报道的紧密类似的共生梭菌谷氨酸脱氢酶的酶 - 辅酶和酶 - L - 谷氨酸复合物的晶体结构研究（Stillman, T. J., Baker, P. J., Britton, K. L., and Rice, D. W. (1993) J. Mol. Biol. 234, 1131 - 1139）为该现象提供了物理解释的基础。我们得出结论，电荷转移现象是由底物未质子化的α - 氨基与酶的一种形式紧密并置引起的，在这种酶形式中，构象变化导致活性位点裂隙完全关闭。
