1cij

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spinqualitylabelsall models 1cij, resolution 2.30Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

HALOALKANE DEHALOGENASE SOAKED WITH HIGH CONCENTRATION OF BROMIDE

Overview

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.

About this Structure

1CIJ is a Single protein structure of sequence from Xanthobacter autotrophicus with BR as ligand. Active as Haloalkane dehalogenase, with EC number 3.8.1.5 Full crystallographic information is available from OCA.

Reference

Crystallographic and kinetic evidence of a collision complex formed during halide import in haloalkane dehalogenase., Pikkemaat MG, Ridder IS, Rozeboom HJ, Kalk KH, Dijkstra BW, Janssen DB, Biochemistry. 1999 Sep 14;38(37):12052-61. PMID:10508409

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