Crystallographic and kinetic evidence of a collision complex formed duringhalide import in haloalkane dehalogenase

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 an alyzed by measuring halide binding with stopped-flow fluorescence experimen ts. At high halide concentrations, halide import occurs predominantly via t he rapid formation of a weak initial collision complex, followed by transpo rt of the ion to the active site. To obtain mole insight in this collision complex, we determined the X-ray structure of DhlA in the presence of bromi de and investigated the kinetics of mutants that were constructed on the ba sis 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 o f the enzyme. One of the weakly bound ions is close to Thr197 and Phe294, n ear the entrance of the earlier proposed tunnel for substrate import. Kinet ic analysis of bromide import by the Thr197Ala and Phe294Ala mutants of Dhl A at high halide concentration showed that the rate constants for halide bi nding no longer displayed a wild-type-like parabolic increase with increasi ng bromide concentrations. This is in agreement with an elimination or a de crease in affinity of the surface-located halide-binding site. Likewise, ch loride binding kinetics of the mutants indicated significant differences wi th wild-type enzyme. The results: indicate that Thr197 and Phe294 are invol ved in the formation of an initial collision complex for halide import in D hlA and provide experimental evidence for the role of the tunnel in substra te and product transport.