KINETICS OF HALIDE RELEASE OF HALOALKANE DEHALOGENASE - EVIDENCE FOR A SLOW CONFORMATIONAL CHANGE

Haloalkane dehalogenase converts haloalkanes to their corresponding al cohols and halides, The reaction mechanism involves the formation of a covalent alkyl-enzyme complex which is hydrolyzed by water. The activ e site is a hydrophobic cavity buried between the main domain and the cap domain of the enzyme. The enzyme has a broad substrate specificity , but the k(cat) values of the enzyme for the best substrates 1,2-dich loroethane and 1,2-dibromoethane are rather low (3 and 3.5 s(-1), resp ectively). Stopped-flow fluorescence experiments with substrate under single-turnover conditions indicated that halide release could limit t he overall k(cat). Furthermore, at 5 mM 1,2-dibromoethane the observed rate of substrate binding to free enzyme was faster than 700 s(-1) (w ithin the dead time of the stopped-flow instrument) whereas displaceme nt of halide by 5 mM 1,2-dibromoethane occurred at a rate of only 8 s( -1). The binding of bromide and chloride to free enzyme was also studi ed using stopped-flow fluorescence, and the dependence of k(obs) on th e halide concentration suggested that there were two parallel routes f or halide binding. One route, in which a slow enzyme isomerization is followed by rapid halide binding, was predominant at low halide concen trations. The other route involves rapid binding into an initial colli sion complex followed by a slow enzyme isomerization step and prevaile d at higher halide concentrations. The overall rate of halide release was low and limited by a slow enzyme isomerization preceding actual re lease (9 and 14.5 s(-1) for bromide and chloride, respectively). We pr opose that this slow isomerization is a conformational change in the c ap domain that is necessary to allow water to enter and solvate the ha lide ion. A solvent kinetic isotope effect of (H2O)-H-2 was found both on k(cat) and on the rate of halide release. (H2O)-H-2 mainly affecte d the rate of the conformational change, which is in agreement with th is step being rate-limiting and the overall stabilizing effect of (H2O )-H-2 on the conformation of proteins.