title: A pre-steady state kinetic analysis of the αY60W mutant of trans-3-chloroacrylic acid dehalogenase: implications for the mechanism of the wild-type enzyme.
abstract: The bacterial degradation of the nematicide 1,3-dichloropropene, an isomeric mixture, requires the action of trans- and cis-3-chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively). Both enzymes are tautomerase superfamily members and share a core catalytic mechanism for the hydrolytic dehalogenation of the respective isomer of 3-haloacrylate. The observation that cis-CaaD requires two additional residues raises the question of how CaaD conducts a comparable reaction with fewer catalytic residues. As part of an effort to determine the basis for the apparently simpler CaaD-catalyzed reaction, the kinetic mechanism was determined by stopped-flow and chemical-quench techniques using a fluorescent mutant form of the enzyme, αY60W-CaaD, and trans-3-bromoacrylate as the substrate. The data from these experiments as well as bromide inhibition studies are best accommodated by a six-step model that provides individual rate constants for substrate binding, chemistry, and a proposed conformational change occurring after chemistry followed by release of malonate semialdehyde and bromide. The conformational change and product release rates are comparable, and together they limit the rate of turnover. The kinetic analysis and modeling studies validate the αY60W-CaaD mutant as an accurate reporter of active site events during the course of the enzyme-catalyzed reaction. The kinetic mechanism for the αY60W-CaaD-catalyzed reaction is comparable to that obtained for the cis-CaaD-catalyzed reaction. The kinetic model and the validated αY60W-CaaD mutant set the stage for an analysis of active site mutants to explore the contributions of individual catalytic residues and the basis for the simplicity of the reaction.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, United States.
authors.forename: Jamison P
authors.initials: JP
authors.lastname: Huddleston
authors.forename: Gottfried K
authors.initials: GK
authors.lastname: Schroeder
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Johnson
authors.forename: Christian P
authors.initials: CP
authors.lastname: Whitman
mesh_terms: D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D006867:Hydrolases; C077721:trans-3-chloroacrylic acid dehalogenase