Krooshof G H, Ridder I S, Tepper A W, Vos G J, Rozeboom H J, Kalk K H, Dijkstra B W, Janssen D B
Laboratory of Biochemistry, BIOSON Research Institute, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
Biochemistry. 1998 Oct 27;37(43):15013-23. doi: 10.1021/bi9815187.
Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. Trp175 forms a halogen/halide-binding site in the 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 Km 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 kcat for DBE, which was 2-fold higher than the wild-type kcat. 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 Clalpha-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.
卤代烷脱卤酶（DhlA）通过烷基 - 酶中间体催化卤代烷的水解。色氨酸175（Trp175）与色氨酸125（Trp125）一起在活性位点腔中形成一个卤素/卤化物结合位点。为了更深入了解Trp175在DhlA中的作用，我们对175位残基进行了突变，并研究了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的催化性能很重要。对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Km增加6倍是由于DBE结合速率和碳 - 溴键断裂速率均降低所致。此外，突变酶中溴离子释放之前的酶异构化速度加快了4倍。结果，烷基 - 酶中间体的水解速率成为DBE的kcat的主要决定因素，其比野生型kcat高2倍。突变酶在pH 6时的X射线结构表明，酶的主链结构保持完整，酪氨酸侧链与野生型酶中Trp175处于同一平面。Tyr175和Trp125的稳定α-碳原子的芳香环相距0.7埃，并且由于突变残基的尺寸较小，腔的体积增加了五分之一。突变酶和野生型酶在pH 5时的X射线结构表明，乙酸分子结合后Tyr175侧链发生旋转，仅留下其一个氧原子与Trp125的吲哚氮形成氢键。这些结构变化表明175位残基与活性位点中的卤素原子或卤离子之间的相互作用减弱，有助于解释由Trp175Tyr突变引起的动力学变化。
