Release of Halide Ions from the Buried Active Site of the Haloalkane Dehalogenase LinB Revealed by Stopped-Flow Fluorescence Analysis and Free Energy Calculations
Hladilkova, J., Prokop, Z., Chaloupkova, R., Damborsky, J., Jungwirth, P.
JOURNAL OF PHYSICAL CHEMISTRY B 117: 14329-14335 (2013)
Release of halide ions is an essential step of the catalytic cycle of haloalkane dehalogenases. Here we describe experimentally and computationally the process of release of a halide anion from the buried active site of the haloalkane dehalogenase LinB. Using stopped-flow fluorescence analysis and umbrella sampling free energy calculations we showed that the anion binding is ion-specific and follows the ordering I– > Br– > Cl–. We have also addressed the issue of the protonation state of the catalytic His272 residue and its effect on the process of halide release. While deprotonation of His272 increases binding of anions in the access tunnel, we showed that the anionic ordering does not change with the switch of the protonation state. We also demonstrated that a sodium cation could relatively easily enter the active site, provided the His272 residue is singly protonated, and replace thus the missing proton. In contrast, Na+ is strongly repelled from the active site containing the doubly protonated His272 residue. Our study contributes towards understanding of the reaction mechanism of haloalkane dehalogenase enzyme family. Determination of the protonation state of the catalytic histidine throughout the catalytic cycle remains a challenge for future studies.