Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden.
Biochemistry. 2010 Mar 16;49(10):2297-304. doi: 10.1021/bi902157b.
The underlying enzyme kinetics behind the regioselective promiscuity shown by epoxide hydrolases toward certain epoxides has been studied. The effects of temperature and pH on regioselectivity were investigated by analyzing the stereochemistry of hydrolysis products of (1R,2R)-trans-2-methylstyrene oxide between 14-46 degrees C and pH 6.0-9.0, either catalyzed by the potato epoxide hydrolase StEH1 or in the absence of enzyme. In the enzyme-catalyzed reaction, a switch of preferred epoxide carbon that is subjected to nucleophilic attack is observed at pH values above 8. The enzyme also displays cooperativity in substrate saturation plots when assayed at temperatures < or = 30 degrees C and at intermediate pH. The cooperativity is lost at higher assay temperatures. Cooperativity can originate from a kinetic mechanism involving hysteresis and will be dependent on the relationship between k(cat) and the rate of interconversion between two different Michaelis complexes. In the case of the studied reactions, the proposed different Michaelis complexes are enzyme-substrate complexes in which the epoxide substrate is bound in different binding modes, allowing for separate pathways toward product formation. The assumption of separated, but interacting, reaction pathways is supported by that formation of the two product enantiomers also displays distinct pH dependencies of k(cat)/K(M). The thermodynamic parameters describing the differences in activation enthalpy and entropy suggest that (1) regioselectivity is primarily dictated by differences in activation entropy with positive values of both DeltaDeltaH(++) and DeltaDeltaS(++) and (2) the hysteretic behavior is linked to an interconversion between Michaelis complexes with rates increasing with temperature. From the collected data, we propose that hysteresis, regioselectivity, and, when applicable, hysteretic cooperativity are closely linked properties, explained by the kinetic mechanism earlier introduced by our group.
已研究了环氧化物水解酶对某些环氧化物表现出的区域选择性混杂背后的潜在酶动力学。通过分析（1R，2R）-反式-2-甲基苯乙烯氧化物在 14-46°C 和 pH 6.0-9.0 之间的水解产物的立体化学，研究了温度和 pH 对区域选择性的影响，这些反应要么由马铃薯环氧化物水解酶 StEH1 催化，要么在没有酶的情况下进行。在酶催化反应中，在 pH 值高于 8 时观察到受亲核攻击的优选环氧化物碳的开关。当在温度 <或= 30°C 和中间 pH 下进行测定时，该酶在底物饱和图中还显示出协同作用。在较高的测定温度下，协同作用会丢失。协同作用可能源于涉及滞后的动力学机制，并且将取决于 k(cat)与两种不同米氏复合物之间的转换速率之间的关系。在所研究的反应中，所提出的不同米氏复合物是酶-底物复合物，其中环氧化物底物以不同的结合方式结合，允许形成两种不同的产物形成途径。假设存在分离但相互作用的反应途径得到了以下事实的支持：两种产物对映体的形成也显示出 k(cat)/K(M)的独特 pH 依赖性。描述活化焓和熵差异的热力学参数表明，（1）区域选择性主要由活化熵的差异决定，正的 DeltaDeltaH(++)和 DeltaDeltaS(++)值，（2）滞后行为与米氏复合物之间的转换有关，其速率随温度增加而增加。根据收集到的数据，我们提出滞后性、区域选择性以及在适用情况下的滞后协同作用是密切相关的特性，这可以通过我们小组早些时候提出的动力学机制来解释。
