Kinetic analysis and X-ray structure of haloalkane dehalogenase with a modified halide-binding site
Kinetic analysis and X-ray structure of haloalkane dehalogenase with a modified halide-binding site
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DOI：
10.1021/bi9815187
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发表时间：
1998-10-27
期刊：
影响因子：
2.9
通讯作者：
Janssen, DB
中科院分区：
文献类型：
--
作者：
Krooshof, GH;Ridder, IS;Janssen, DB
卤代烷脱卤酶(DhlA)通过一种烷基酶中间体催化卤代烃的水解。Trp175与Trp125一起在活性中心腔内形成卤素/卤化物结合部位。为了更深入地了解Trp175在DhlA中的作用，我们突变了175个残基，并探索了Trp175Tyr酶的动力学和X射线结构。诱变研究表明，175位的芳香族残基对DhlA的催化性能有重要影响。Trp175Tyr-DhlA的稳态前动力学分析表明，观察到的1，2-二溴乙烷(DBE)的K-m增加6倍是由于DBE结合速度和碳-溴键断裂速度的降低。此外，在突变酶中，溴化物释放之前的酶异构化速度提高了4倍。结果，烷基酶中间体的水解率成为决定DBE的k(CAT)的主要因素，是野生型k(CAT)的2倍。突变酶在pH值为6时的X射线结构表明，酶的骨架结构保持完整，酪氨酸侧链与野生型酶中的Trp175位于同一平面。Tyr175和Trp125的氯α稳定芳环相距0.7A，由于突变残基的尺寸较小，空腔体积增加了五分之一。突变和野生型酶在pH值为5时的X-射线结构表明，当与醋酸分子结合时，Tyr175侧链发生旋转，只留下一个氧原子氢键与Trp125的吲哚氮结合。这些结构变化表明残基175与活性部位的卤素原子或卤离子之间的相互作用减弱，有助于解释Trp175Tyr突变引起的动力学变化。
Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. Trp175 forms a halogen/halide-binding site in he active-site cavity together with Trp125. To get more insight in the role of Trp175 in DhlA, we mutated residue 175 and explored the kinetics and X-ray structure of the Trp175Tyr enzyme. The mutagenesis study indicated that an aromatic residue at position 175 is important for the catalytic performance of DhlA. Pre-steady-state kinetic analysis of Trp175Tyr-DhlA showed that the observed 6-fold increase of the K-m for 1,2-dibromoethane (DBE) results from reduced rates of both DBE binding and cleavage of the carbon-bromine bond. Furthermore, the enzyme isomerization preceding bromide release became 4-fold faster in the mutant enzyme. As a result, the rate of hydrolysis of the alkyl-enzyme intermediate became the main determinant of the k(cat) for DBE, which was 2-fold higher than the wild-type k(cat). The X-ray structure of the mutant enzyme at pH 6 showed that the backbone structure of the enzyme remains intact and that the tyrosine side chain lies in the same plane as Trp175 in the wild-type enzyme. The Cl alpha-stabilizing aromatic rings of Tyr175 and Trp125 are 0.7 A further apart and due to the smaller size of the mutated residue, the volume of the cavity has increased by one-fifth. X-ray structures of mutant and wild-type enzyme at pH 5 demonstrated that the Tyr175 side chain rotated away upon binding of an acetic acid molecule, leaving one of its oxygen atoms hydrogen bonded to the indole nitrogen of Trp125 only. These structural changes indicate a weakened interaction between residue 175 and the halogen atom or halide ion in the active site and help to explain the kinetic changes induced by the Trp175Tyr mutation.