Catalytic cycle of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus. Solvent viscosity, deuterium isotope effects, and proton inventory studies.
Catalytic cycle of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus. Solvent viscosity, deuterium isotope effects, and proton inventory studies.
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来自蜡样芽胞杆菌的磷脂酰胆碱偏好型磷脂酶 C 的催化循环。
DOI：
10.1021/bi9821216
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Hergenrother,PJ
中科院分区：
文献类型：
--
作者：
Martin,SF;Hergenrother,PJ
蜡状芽孢杆菌磷脂酶C(PLCBc)是一种大小为28.5 kDa的酶，其活性部位含有3个锌离子。虽然人们对PLCBC活性中心氨基酸在结合和催化中所起的作用已知很多，但对于PLCBC催化磷脂水解的速率决定步骤和酶的催化循环却知之甚少。为了深入了解水解的这些方面，进行了溶剂粘度变化实验，以确定外部步骤(底物结合或产物释放)或内部步骤(水解)是限速的。数据表明，PLCBc催化的反应不受溶剂粘度变化的影响。这一观察结果与底物结合或产物释放是速率决定的概念不一致，并支持化学步骤是速率限制的假设。此外，氘同位素效应为1.9和线性质子库存图表明，在速率决定步骤中转移了一个质子。这些数据可能被用来制定一个全面的催化循环，这是第一次基于实验证据。在这个机制中，PLCBC的Asp55激活了一个活性部位的水分子，以攻击磷酸二酯键，该键的水解是限速的。磷酰胆碱产物是第一个离开活性部位的，其次是二酰甘油。
The phosphatidylcholine-preferring phospholipase C fromBacillus cereus(PLCBc) is a 28.5 kDa enzyme with three zinc ions in its active site. Although much is known about the roles that various PLCBcactive site amino acids play in binding and catalysis, there is little information about the rate-determining step of the PLCBc-catalyzed hydrolysis of phospholipids and the catalytic cycle of the enzyme. To gain insight into these aspects of the hydrolysis, solvent viscosity variation experiments were conducted to determine whether an external step (substrate binding or product release) or an internal step (hydrolysis) is rate-limiting. The data indicate that the PLCBc-catalyzed reaction is unaffected by changes in solvent viscosity. This observation is inconsistent with the notion of substrate binding or product release being rate-determining and supports the hypothesis that a chemical step is rate-limiting. Furthermore, a deuterium isotope effect of 1.9 and a linear proton inventory plot indicate one proton is transferred in the rate-determining step. These data may be used to formulate a comprehensive catalytic cycle that is for the first time based on experimental evidence. In this mechanism, Asp55 of PLCBcactivates an active site water molecule for attack on the phosphodiester bond, the hydrolysis of which is rate-limiting. The phosphorylcholine product is the first to leave the active site, followed by diacylglycerol.