Kinetic Characterization and X-ray Structure of a Mutant of Haloalkane Dehalogenase with Higher Catalytic Activity and Modified Substrate Range,

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Abstract
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 Clβ 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.