Pikkemaat M G, Ridder I S, 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. 1999 Sep 14;38(37):12052-61. doi: 10.1021/bi990849w.
Haloalkane dehalogenase (DhlA) converts haloalkanes to their corresponding alcohols and halide ions. The rate-limiting step in the reaction of DhlA is the release of the halide ion. The kinetics of halide release have been analyzed by measuring halide binding with stopped-flow fluorescence experiments. At high halide concentrations, halide import occurs predominantly via the rapid formation of a weak initial collision complex, followed by transport of the ion to the active site. To obtain more insight in this collision complex, we determined the X-ray structure of DhlA in the presence of bromide and investigated the kinetics of mutants that were constructed on the basis of this structure. The X-ray structure revealed one bromide ion firmly bound in the active site and two bromide ions weakly bound on the surface of the enzyme. One of the weakly bound ions is close to Thr197 and Phe294, near the entrance of the earlier proposed tunnel for substrate import. Kinetic analysis of bromide import by the Thr197Ala and Phe294Ala mutants of DhlA at high halide concentration showed that the rate constants for halide binding no longer displayed a wild-type-like parabolic increase with increasing bromide concentrations. This is in agreement with an elimination or a decrease in affinity of the surface-located halide-binding site. Likewise, chloride binding kinetics of the mutants indicated significant differences with wild-type enzyme. The results indicate that Thr197 and Phe294 are involved in the formation of an initial collision complex for halide import in DhlA and provide experimental evidence for the role of the tunnel in substrate and product transport.
卤代烷脱卤酶（DhlA）可将卤代烷转化为相应的醇和卤离子。DhlA反应中的限速步骤是卤离子的释放。通过使用停流荧光实验测量卤离子结合情况，分析了卤离子释放的动力学。在高卤离子浓度下，卤离子的导入主要通过快速形成弱初始碰撞复合物，随后离子转运至活性位点。为了更深入了解这种碰撞复合物，我们确定了存在溴离子时DhlA的X射线结构，并研究了基于该结构构建的突变体的动力学。X射线结构显示一个溴离子牢固地结合在活性位点，两个溴离子弱结合在酶的表面。其中一个弱结合离子靠近Thr197和Phe294，位于先前提出的底物导入通道入口附近。对DhlA的Thr197Ala和Phe294Ala突变体在高卤离子浓度下溴离子导入的动力学分析表明，随着溴离子浓度增加，卤离子结合的速率常数不再呈现野生型样的抛物线型增加。这与表面定位的卤离子结合位点亲和力的消除或降低一致。同样，突变体的氯离子结合动力学表明与野生型酶存在显著差异。结果表明，Thr197和Phe294参与了DhlA中卤离子导入初始碰撞复合物的形成，并为通道在底物和产物转运中的作用提供了实验证据。
