Kinetic characterization and X-ray structure of a mutant of haloalkane dehalogenase with higher catalytic activity and modified substrate range

Biochemistry. 1996 Oct 8;35(40):13186-95. doi: 10.1021/bi961151a.


Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helix-loop-helix structure that covers the active site cavity of the enzyme, interacts with the C1 beta of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher Kcat/Km for 1-chlorohexane and a 2-fold higher Kcat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in Kcat found for 1,2-dibromoethane conversion could be attributed to an increase in the rate of an enzyme isomerization step that preceeds halide release. The observed conformational difference between the helix-loop-helix structures of the wild-type enzyme and the faster mutant suggests that the isomerization required for halide release could be a conformational change that takes place in this region of the cap domain of the dehalogenase. It is proposed that Phe172 is involved in stabilization of the helix-loop-helix structure that covers the active site of the enzyme and creates a rigid hydrophobic cavity for small apolar halogenated alkanes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / genetics
  • Amino Acids / metabolism
  • Bacteria / enzymology
  • Bromides / metabolism
  • Chlorides / metabolism
  • Crystallography, X-Ray
  • Ethylene Dibromide / metabolism
  • Helix-Loop-Helix Motifs
  • Hexanes / metabolism
  • Hydrocarbons, Chlorinated / metabolism
  • Hydrolases / chemistry*
  • Hydrolases / genetics
  • Hydrolases / metabolism*
  • Kinetics
  • Models, Molecular
  • Molecular Structure
  • Mutagenesis, Site-Directed
  • Phenylalanine / genetics
  • Phenylalanine / metabolism
  • Protein Binding
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Substrate Specificity


  • Amino Acids
  • Bromides
  • Chlorides
  • Hexanes
  • Hydrocarbons, Chlorinated
  • Recombinant Proteins
  • Ethylene Dibromide
  • Phenylalanine
  • Hydrolases
  • haloalkane dehalogenase
  • 1-chlorohexane