Schopfer L M, Massey V, Ghisla S, Thorpe C
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Biochemistry. 1988 Aug 23;27(17):6599-611. doi: 10.1021/bi00417a059.
Pig kidney general acyl-CoA dehydrogenase (GAD) can be reduced by butyryl-CoA to form reduced enzyme and crotonyl-CoA. This reaction is reversible. Stopped-flow, kinetic investigations on GAD have been made, using the following reaction pairs: oxidized GAD/butyryl-CoA, oxidized GAD/crotonyl-CoA, oxidized GAD/alpha,beta-dideuteriobutyryl-CoA, reduced GAD/butyryl-CoA, and reduced GAD/crotonyl-CoA (in 50 mM potassium phosphate buffer, pH 7.6 at 4 degrees C). Reduction of GAD by butyryl-CoA is triphasic. The slowest phase is 100-fold slower than the preceding phase and appears to represent a secondary process not directly related to the primary reduction events. The first two fast phases are responsible for reduction of GAD. Reduction proceeds via a reduced enzyme/crotonyl-CoA charge-transfer complex. alpha, beta-Dideuteriobutyryl-CoA elicits a major deuterium isotope effect (15-fold) on the reduction reaction. Oxidation of GAD by crotonyl-CoA is biphasic. Oxidation proceeds via the same reduced enzyme/crotonyl-CoA charge-transfer complex seen during reduction. The oxidation reaction ends in a mixture composed largely of oxidized GAD species. From the data, we constructed a mechanism for the reduction/oxidation of GAD by butyryl-CoA/crotonyl-CoA. This mechanism was then used to simulate all of the observed kinetic time course data, using a single set of kinetic parameters. A close correspondence between the observed and simulated data was obtained.
猪肾通用酰基辅酶A脱氢酶（GAD）可被丁酰辅酶A还原，形成还原型酶和巴豆酰辅酶A。此反应是可逆的。利用以下反应对，对GAD进行了停流动力学研究：氧化型GAD/丁酰辅酶A、氧化型GAD/巴豆酰辅酶A、氧化型GAD/α,β-二氘代丁酰辅酶A、还原型GAD/丁酰辅酶A和还原型GAD/巴豆酰辅酶A（在50 mM磷酸钾缓冲液中，4℃，pH 7.6）。丁酰辅酶A对GAD的还原是三相的。最慢的相比前一相慢100倍，似乎代表了一个与初级还原事件无直接关系的次级过程。前两个快速相负责GAD的还原。还原通过还原型酶/巴豆酰辅酶A电荷转移复合物进行。α,β-二氘代丁酰辅酶A对还原反应产生了主要的氘同位素效应（15倍）。巴豆酰辅酶A对GAD的氧化是双相的。氧化通过还原过程中所见的相同还原型酶/巴豆酰辅酶A电荷转移复合物进行。氧化反应以主要由氧化型GAD物种组成的混合物结束。根据这些数据，我们构建了丁酰辅酶A/巴豆酰辅酶A对GAD还原/氧化的机制。然后使用一组动力学参数，用该机制模拟了所有观察到的动力学时间进程数据。观察到的数据与模拟数据之间获得了密切的对应关系。
