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A pre-steady state kinetic analysis of the ?Y60W 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, ?Y60W-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 ?Y60W-CaaD mutant as an accurate reporter of active site events during the course of the enzyme-catalyzed reaction. The kinetic mechanism for the ?Y60W-CaaD-catalyzed reaction is comparable to that obtained for the cis-CaaD-catalyzed reaction. The kinetic model and the validated ?Y60W-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.

SUBMITTER: Huddleston JP

PROVIDER: S-EPMC3518313 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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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.

Huddleston Jamison P JP Schroeder Gottfried K GK Johnson Kenneth A KA Whitman Christian P CP

Biochemistry 20121108 46

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 cataly ...[more]

PMID: 23110338

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Project description:Isomer-specific 3-chloroacrylic acid dehalogenases catalyze the hydrolytic dehalogenation of the cis- and trans-isomers of 3-chloroacrylate to yield malonate semialdehyde. These reactions represent key steps in the degradation of the nematocide, 1,3-dichloropropene. The kinetic mechanism of cis-3-chloroacrylic acid dehalogenase (cis-CaaD) has now been examined using stopped-flow and chemical-quench techniques. Stopped-flow analysis of the reaction, following the fluorescence of an active site tryptophan, is consistent with a minimal three-step model involving substrate binding, chemistry, and product release. Chemical-quench experiments show burst kinetics, indicating that product release is at least partially rate limiting. Global fitting of all of the kinetic results by simulation is best accommodated by a four-step mechanism. In the final kinetic model, the enzyme binds substrate with an immediate isomerization to an alternate fluorescent form and chemistry occurs, followed by the ordered release of two products, with the release of the first product as the rate-limiting step. Bromide ion is a competitive inhibitor of the reaction indicating that it binds to the free enzyme rather than to the enzyme with one product still bound. This observation suggests that malonate semialdehyde is the first product released by the enzyme (rate limiting), followed by halide. A comparison of the unliganded cis-CaaD crystal structure with that of an inactivated cis-CaaD where the prolyl nitrogen of Pro-1 is covalently attached to (R)-2-hydroxypropanoate provides a possible explanation for the isomerization step. The structure of the covalently modified enzyme shows that a seven-residue loop comprised of residues 32-38 is closed down on the active site cavity where the backbone amides of two residues (Phe-37 and Leu-38) interact with the carboxylate group of the adduct. In the unliganded form, the same loop points away from the active site cavity. Similarly, substrate binding may cause this loop to close down on the active site and sequester the reaction from the external environment.

Dataset Information

A pre-steady state kinetic analysis of the ?Y60W mutant of trans-3-chloroacrylic acid dehalogenase: implications for the mechanism of the wild-type enzyme.

Publications

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.

Similar Datasets

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