Tan Xiangshi, Surovtsev Ivan V, Lindahl Paul A
Department of Chemistry, Texas A&M University, College Station, Texas 77843, USA.
J Am Chem Soc. 2006 Sep 20;128(37):12331-8. doi: 10.1021/ja0627702.
Acetyl-CoA synthase/carbon monoxide dehydrogenase is a Ni-Fe-S-containing enzyme that catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group. The methyl group is transferred onto the enzyme from a corrinoid-iron-sulfur protein (CoFeSP). The kinetics of two steps within the catalytic mechanism were studied using the stopped-flow method, including the insertion of CO into a putative Ni(2+)-CH(3) bond and the transfer of the resulting acetyl group to CoA. Neither step had been studied previously. Reactions were monitored indirectly, starting with the methylated intermediate form of the enzyme. Resulting traces were analyzed by constructing a simple kinetic model describing the catalytic mechanism under reducing conditions. Besides methyl group transfer, CO insertion, and acetyl group transfer, fitting to experimental traces required the inclusion of an inhibitory step in which CO reversibly bound to the form of the enzyme obtained immediately after product release. Global simulation of the reported datasets afforded a consistent set of kinetic parameters. The equilibrium constant for the overall synthesis of acetyl-CoA was estimated and compared to the product of the individual equilibrium constants. Simulations obtained with the model duplicated the essential behavior of the enzyme, in terms of the variation of activity with [CO], and the time-dependent decay of the NiFeC EPR signal upon reaction with CoFeSP. Under standard assay conditions, the model suggests that the vast majority of active enzyme molecules in a population should be in the methylated form, suggesting that the subsequent catalytic step, namely CO insertion, is rate limiting. This conclusion is further supported by a sensitivity analysis showing that the rate is most sensitively affected by a change in the rate coefficient associated with the CO insertion step.
乙酰辅酶A合成酶/一氧化碳脱氢酶是一种含镍铁硫的酶，它催化由一氧化碳、辅酶A和一个甲基合成乙酰辅酶A。甲基从类咕啉铁硫蛋白（CoFeSP）转移到该酶上。使用停流法研究了催化机制中两个步骤的动力学，包括一氧化碳插入假定的Ni(2+)-CH(3)键以及将生成的乙酰基转移到辅酶A上。这两个步骤此前均未被研究过。反应从酶的甲基化中间形式开始进行间接监测。通过构建一个描述还原条件下催化机制的简单动力学模型来分析所得的曲线。除了甲基转移、一氧化碳插入和乙酰基转移外，为了拟合实验曲线，还需要纳入一个抑制步骤，即一氧化碳与产物释放后立即得到的酶形式可逆结合。对所报道数据集的整体模拟得到了一组一致的动力学参数。估计了乙酰辅酶A整体合成的平衡常数，并与各个平衡常数的乘积进行了比较。用该模型进行的模拟重现了该酶的基本行为，包括活性随[CO]的变化以及与CoFeSP反应后NiFeC EPR信号随时间的衰减。在标准测定条件下，该模型表明群体中绝大多数活性酶分子应为甲基化形式，这表明随后的催化步骤，即一氧化碳插入，是限速步骤。敏感性分析进一步支持了这一结论，该分析表明速率最敏感地受到与一氧化碳插入步骤相关的速率系数变化的影响。
