Rink R, Janssen D B
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
Biochemistry. 1998 Dec 22;37(51):18119-27. doi: 10.1021/bi9817257.
Epoxide hydrolase from Agrobacterium radiobacter AD1 catalyzes the enantioselective hydrolysis of styrene oxide with an E value of 16. The (R)-enantiomer of styrene oxide is first converted with a k(cat) of 3.8 s(-1), and the conversion of the (S)-enantiomer is inhibited. The latter is subsequently hydrolyzed with a k(cat) of 10.5 s(-1). The pre-steady-state kinetic parameters were determined for both enantiomers with stopped-flow fluorescence and rapid-quench techniques. For (R)-styrene oxide a four-step mechanism was needed to describe the data. It involved the formation of a Michaelis complex that is in rapid equilibrium with free enzyme and substrate, followed by rapid and reversible alkylation of the enzyme. A unimolecular isomerization of the alkylated enzyme precedes the hydrolysis of the covalent intermediate, which could be observed due to an enhancement of the intrinsic protein fluorescence during this step. The conversion of (S)-styrene oxide could be described by a three-step mechanism, which also involved reversible and rapid formation of an ester intermediate from a Michaelis complex and its subsequent slow hydrolysis as the rate-limiting step. The unimolecular isomerization step has not been observed for rat microsomal epoxide hydrolase, for which a kinetic mechanism was recently established [Tzeng, H.-F., Laughlin, L. T., Lin, S., and Armstrong, R. N. (1996) J. Am. Chem. Soc. 118, 9436-9437]. For both enantiomers of styrene oxide, the Km value was much lower than the substrate binding constant K(S) due to extensive accumulation of the covalent intermediate. The enantioselectivity was more pronounced in the alkylation rates than in the rate-limiting hydrolysis steps. The combined reaction schemes for (R)- and (S)-styrene oxide gave an accurate description of the epoxide hydrolase catalyzed kinetic resolution of racemic styrene oxide.
来自放射形土壤杆菌AD1的环氧水解酶催化环氧苯乙烷的对映选择性水解，E值为16。环氧苯乙烷的（R）-对映体首先以3.8 s⁻¹的催化常数（k(cat)）进行转化，而（S）-对映体的转化受到抑制。后者随后以10.5 s⁻¹的催化常数进行水解。采用停流荧光法和快速淬灭技术测定了两种对映体的预稳态动力学参数。对于（R）-环氧苯乙烷，需要一个四步机制来描述数据。它涉及形成一个与游离酶和底物处于快速平衡的米氏复合物，随后酶进行快速且可逆的烷基化。烷基化酶的单分子异构化先于共价中间体的水解，这一步骤可通过蛋白质固有荧光增强观察到。（S）-环氧苯乙烷的转化可用一个三步机制来描述，该机制也涉及从米氏复合物可逆且快速地形成酯中间体，以及其随后缓慢水解作为限速步骤。大鼠微粒体环氧水解酶未观察到单分子异构化步骤，最近已为其建立了动力学机制 [曾，H.-F.，劳克林，L. T.，林，S.，和阿姆斯特朗，R. N.（1996年）《美国化学会志》118，9436 - 9437]。对于环氧苯乙烷的两种对映体，由于共价中间体的大量积累，米氏常数（Km）值远低于底物结合常数K(S)。对映选择性在烷基化速率中比在限速水解步骤中更明显。（R）-和（S）-环氧苯乙烷的联合反应方案准确描述了环氧水解酶催化外消旋环氧苯乙烷的动力学拆分。
