title: Stereospecific alkylation of cis-3-chloroacrylic acid dehalogenase by (R)-oxirane-2-carboxylate: analysis and mechanistic implications.
abstract: The enzymes trans-3-chloroacrylic acid dehalogenase (CaaD) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) represent the two major classes of bacterial, isomer-selective 3-chloroacrylic acid dehalogenases. They catalyze the hydrolytic dehalogenation of either trans- or cis-3-haloacrylates to yield malonate semialdehyde, presumably through unstable halohydrin intermediates. In view of a proposed general acid/base mechanism for these enzymes, (R)- and (S)-oxirane-2-carboxylate were investigated as potential irreversible inhibitors. Only cis-CaaD is irreversibly inhibited in a time- and concentration-dependent manner and only by the (R)-enantiomer of oxirane-2-carboxylate. The enzyme displays saturation kinetics and is protected from inactivation by the presence of substrate. These findings indicate that the inactivation process involves the initial formation of a reversibly bound enzyme-inhibitor complex at the active site followed by covalent modification. Mass spectral analysis of the inactivated cis-CaaD shows that Pro-1 is the site of modification. It has also been determined that Arg-70 and Arg-73 are required for covalent modification because incubation of either the R70A or R73A mutant with inhibitor does not result in enzyme alkylation. Studies of the pH dependence of the kinetic parameters of wild-type cis-CaaD reveal that a protonated group with a pK(a) of approximately 9.3 is essential for catalysis. The group is likely Pro-1, making it predominately a charged species under the conditions of the inactivation experiments. Two mechanisms could account for these observations. In one mechanism, the oxirane undergoes acid-catalyzed ring opening followed by alkylation of the conjugate base of Pro-1. Alternatively, the oxirane undergoes a nucleophilic substitution reaction where the conjugate base of Pro-1 functions as the nucleophile and an acid catalyst polarizes the carbon oxygen bond. The two arginine residues likely bind the carboxylate group and position the inhibitor in a favorable orientation for the alkylation reaction. These findings set the stage for a crystallographic analysis of the inactived enzyme to delineate further the roles of active site residues in both the inactivation process and the catalytic mechanism.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712-1074, USA.
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Poelarends
authors.forename: Hector
authors.initials: H
authors.lastname: Serrano
authors.forename: William H
authors.initials: WH
authors.lastname: Johnson
authors.forename: Christian P
authors.initials: CP
authors.lastname: Whitman
mesh_terms: D000179:Acrylates / Q000378:metabolism; D000478:Alkylation; D001120:Arginine; D001665:Binding Sites; D002264:Carboxylic Acids / Q000378:metabolism; D002384:Catalysis; D005027:Ethylene Oxide / Q000737:chemistry / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008314:Malonates / Q000378:metabolism; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011549:Pseudomonas / Q000201:enzymology*; D013237:Stereoisomerism
chemical_list: D000179:Acrylates; D002264:Carboxylic Acids; D008314:Malonates; D001120:Arginine; D006867:Hydrolases; C077719:cis-3-chloroacrylic acid dehalogenase; C077721:trans-3-chloroacrylic acid dehalogenase; D005027:Ethylene Oxide