Catalytic mechanism of Escherichia coli alkaline phosphatase: resolution of three variants of the acyl-enzyme mechanism.
Catalytic mechanism of Escherichia coli alkaline phosphatase: resolution of three variants of the acyl-enzyme mechanism.
复制标题
大肠杆菌碱性磷酸酶的催化机制：酰基酶机制的三种变体的解析。
作者：
W. Bloch;M. Gorby
从理论上比较了三种经典的酰基酶机制与大肠杆菌碱性磷酸酶水解底物的预测瞬态动力学。在这三种情况下，酰基酶水解被认为最初主要以与酸产物无机磷酸盐的非共价复合物的形式存在。在一种机制中，预稳态速率控制步骤被认为是酸产物与酶的初始配合物的解离。在另外两种情况下，预稳态速率控制被分配给在底物与游离酶结合之前或之后发生的酶异构化。在过量底物和酸产物的浓度条件下，采用综合速率定律来排除磷酸盐解离和底物结合之间的酶异构化控制预稳态速率的可能性。虽然该机制预测了底物和酸产物之间的预稳态非竞争关系，但4-甲基伞草酰磷酸水解的停止流动动力学表明了一种竞争关系，与其他两种机制中的任何一种一致。在化学计量学限制底物的浓度条件下，计算机模拟消除了底物结合后酶异构化控制速率的可能性。该机制预测底物水解的一级速率常数依赖于底物浓度，这与先前发表的数据不一致[Halford， s.e. (1971) Biochem]。[j]；另外两种机理与实验结果一致。两种浓度条件下的瞬态动力学理论与实验结果的对比表明，大肠杆菌碱性磷酸盐水解磷酸酯的速率控制步骤是“粘性”酸产物与酶的非共价配合物的解离。这一机制解释了在停止流动的动力学痕迹中的异常现象，即亚化学计量的酒精产物释放的预稳态爆发。
Three variants of the classical acyl-enzyme mechanism were compared theoretically with respect to the predicted transient kinetics of substrate hydrolysis by Escherichia coli alkaline phosphatase. In all three, acyl-enzyme hydrolysis was assumed to exist initially primarily as a noncovalent complex with the acid product, inorganic phosphate. In one mechanism, the pre-steady-state rate-controlling step was assumed to be the dissociation of acid product from its initial complex with enzyme. In the other two, pre-steady-state rate control was assigned to an enzyme isomerization occurring before or after substrate binding to free enzyme. Under concentration conditions of excess substrate and acid product, integrated rate laws were used to reject the possibility of pre-steady-state rate control by enzyme isomerization between phosphate dissociation and substrate binding. Whereas this mechanism predicts a pre-steady-state noncompetitive relationship between substrate and acid product, the stopped-flow kinetics of 4-methylumbelliferyl phosphate hydrolysis demonstrates a competitive relationship, consistent with either of the other two mechanisms. Under concentration conditions of stoichiometrically limiting substrate, computer simulations eliminated the possibility of rate control by enzyme isomerization after substrate binding. This mechanism predicts a substrate concentration dependence for the apparent first-order rate constant of substrate hydrolysis which disagrees with previously published data [Halford, S. E. (1971) Biochem. J. 125, 319--327]; the other two mechanisms are consistent with experiment. Comparison of transient kinetic theory and experiment under these two contrasting concentration conditions suggests strongly that the rate-controlling step in phosphate ester hydrolysis by E. coli alkaline phosphate is the dissociation of "sticky" acid product from its noncovalent complex with enzyme. This mechanism explains an anomaly in the stopped-flow kinetic trace, a substoichiometric pre-steady-state burst of alcohol product release.