Kennes C, Pries F, Krooshof G H, Bokma E, Kingma J, Janssen D B
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
Eur J Biochem. 1995 Mar 1;228(2):403-7.
Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in short-chain haloalkanes. Two tryptophan residues of the enzyme (Trp125 and Trp175) form a halide-binding site in the active-site cavity, and were proposed to play a role in catalysis. The function of these residues was studied by replacing Trp125 with phenylalanine, glutamine or arginine and Trp175 by glutamine using site-directed mutagenesis. All mutants except Trp125-->Phe showed a more than 10-fold reduced kcat and much higher Km values with 1,2-dichloroethane and 1,2-dibromoethane than the wild-type enzyme. Fluorescence quenching experiments showed a decrease in the affinity of the mutant enzymes for halide ions. The 2H kinetic isotope effect observed with the wild-type enzyme in deuterium oxide was lost in the active mutants, except the Trp125-->Phe enzyme. The results indicate that both tryptophans are involved in stabilizing the transition state during the nucleophilic substitution reaction that causes carbon-halogen bond cleavage.
卤代烷脱卤酶催化短链卤代烷中碳 - 卤键的水解断裂。该酶的两个色氨酸残基（Trp125和Trp175）在活性位点腔中形成一个卤化物结合位点，并被认为在催化过程中发挥作用。通过定点诱变将Trp125分别替换为苯丙氨酸、谷氨酰胺或精氨酸，将Trp175替换为谷氨酰胺，对这些残基的功能进行了研究。除了Trp125→Phe突变体之外，所有突变体与1,2 - 二氯乙烷和1,2 - 二溴乙烷反应时，其催化常数（kcat）降低了10倍以上，米氏常数（Km）值比野生型酶高得多。荧光猝灭实验表明突变酶对卤离子的亲和力降低。除了Trp125→Phe酶之外，在活性突变体中，野生型酶在重水中观察到的2H动力学同位素效应消失了。结果表明，两个色氨酸都参与了导致碳 - 卤键断裂的亲核取代反应过程中过渡态的稳定。
