Transient kinetic analysis of the catalytic mechanism of haloalkane dehalogenase:Identification of the rate-determining step

We have studied the rate-determining step(s) in the conversion (k(cat)) of 1,2-dibromoethane by haloalkane dehalogenase, a bacterial enzyme involved in degradation of halogenated aliphatic compounds. Analysis of solvent kinetic isotope effects and stopped-flow fluorescence experiments with enzyme under single-turnover conditions indicated that release of the charged bromide ion out of the buried active site cavity could be the slow step in the reaction sequence. Stopped-flow fluorescence analysis of bromide binding and release confirmed this and showed that a slow enzyme-isomerization step preceding the actual bromide release was the slow step that limited the k(cat). We propose that this isomerization is a conformational change of part of the cap domain of the dehalogenase that is necessary to allow water to enter the active site and solvate the charged halide ion.