Institute of Biochemistry, Zhejiang University, Hangzhou, China.
FEBS Lett. 2011 Aug 4;585(15):2545-50. doi: 10.1016/j.febslet.2011.07.006. Epub 2011 Jul 13.
Epoxide hydrolase from Rhodococcus opacus catalyzes the stereospecific hydrolysis of cis-epoxysuccinate to L(+)-tartrate. It shows low but significant similarity to haloacid dehalogenase and haloacetate dehalogenase (16-23% identity). To identify catalytically important residues, we mutated 29 highly conserved charged and polar amino acid residues (except for one alanine). The replacement of D18, D193, R55, K164, H190, T22, Y170, N134 and A188 led to a significant loss in the enzyme activity, indicating their involvement in the catalysis. Single and multiple turnover reaction studies show that the enzyme reaction proceeded through the two-step mechanism involving the formation of a covalent intermediate. We discuss the roles of these residues and propose its possible reaction mechanism.
研究了来源于红平红球菌的环氧化物水解酶（EH）对顺-环氧琥珀酸的立体专一水解作用。该酶与卤代酸脱卤酶（HAD）和卤代乙酸脱卤酶（HAD）具有较低但显著的相似性（16-23%的同源性）。为了确定催化作用的关键残基，我们对 29 个高度保守的带电荷和极性氨基酸残基（除一个丙氨酸外）进行了突变。D18、D193、R55、K164、H190、T22、Y170、N134 和 A188 的取代导致酶活性显著丧失，表明这些残基参与了催化作用。单步和多步反应研究表明，酶反应通过涉及形成共价中间物的两步机制进行。我们讨论了这些残基的作用，并提出了其可能的反应机制。
