Krooshof G H, Kwant E M, Damborský J, Koca J, Janssen D B
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Biochemistry. 1997 Aug 5;36(31):9571-80. doi: 10.1021/bi971014t.
Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289. The role of Asp260, which is the third member of the catalytic triad, was studied by site-directed mutagenesis. Mutation of Asp260 to asparagine resulted in a catalytically inactive D260N mutant, which demonstrates that the triad acid Asp260 is essential for dehalogenase activity. Furthermore, Asp260 has an important structural role, since the D260N enzyme accumulated mainly in inclusion bodies during expression, and neither substrate nor product could bind in the active-site cavity. Activity for brominated substrates was restored to D260N by replacing Asn148 with an aspartic or glutamic acid. Both double mutants D260N+N148D and D260N+N148E had a 10-fold reduced kcat and 40-fold higher Km values for 1,2-dibromoethane compared to the wild-type enzyme. Pre-steady-state kinetic analysis of the D260N+N148E double mutant showed that the decrease in kcat was mainly caused by a 220-fold reduction of the rate of carbon-bromine bond cleavage and a 10-fold decrease in the rate of hydrolysis of the alkyl-enzyme intermediate. On the other hand, bromide was released 12-fold faster and via a different pathway than in the wild-type enzyme. Molecular modeling of the mutant showed that Glu148 indeed could take over the interaction with His289 and that there was a change in charge distribution in the tunnel region that connects the active site with the solvent. On the basis of primary structure similarity between DhlA and other alpha/beta-hydrolase fold dehalogenases, we propose that a conserved acidic residue at the equivalent position of Asn148 in DhlA is the third catalytic triad residue in the latter enzymes.
卤代烷脱卤酶（DhlA）通过烷基 - 酶中间体催化卤代烷的水解反应。共价中间体由天冬氨酸124进行亲核取代反应形成，随后被组氨酸289激活的水分子水解。通过定点诱变研究了催化三联体的第三个成员天冬氨酸260的作用。将天冬氨酸260突变为天冬酰胺会产生催化无活性的D260N突变体，这表明三联体酸天冬氨酸260对脱卤酶活性至关重要。此外，天冬氨酸260具有重要的结构作用，因为D260N酶在表达过程中主要积聚在包涵体中，并且底物和产物都无法结合到活性位点腔中。通过用天冬氨酸或谷氨酸取代天冬酰胺148，D260N对溴化底物的活性得以恢复。与野生型酶相比，双突变体D260N + N148D和D260N + N148E对1,2 - 二溴乙烷的催化常数（kcat）降低了10倍，米氏常数（Km）值提高了40倍。对D260N + N148E双突变体的稳态前动力学分析表明，kcat的降低主要是由于碳 - 溴键断裂速率降低了220倍以及烷基 - 酶中间体水解速率降低了10倍。另一方面，溴离子的释放速度比野生型酶快12倍，且释放途径不同。突变体的分子模型表明，谷氨酸148确实可以取代与组氨酸289的相互作用，并且在连接活性位点与溶剂的通道区域中电荷分布发生了变化。基于DhlA与其他α/β - 水解酶折叠脱卤酶之间的一级结构相似性，我们提出DhlA中天冬酰胺148等效位置的保守酸性残基是后者酶中的第三个催化三联体残基。
