Pre-Steady-State Kinetic Analysis of cis-3-Chloroacrylic Acid Dehalogenase: Analysis and Implications

Abstract

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 and isomerizes to an alternate fluorescent form, chemistry occurs, and is 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 7-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. † This research was supported by the National Institutes of Health Grant GM-65324 and the Robert A. Welch Foundation (F-1334 and SUPPORTING INFORMATION AVAILABLEHere we describe the conventional analysis of the pre-steady state kinetic data (including kinetic schemes and equations used in the fitting process), outline the defining characteristics of the experimental data, evaluate the conventional analysis results, and show why global fitting to a four-step model was required. This is further supported by analysis of the confidence contours and plots for the reversible inhibition of cis-CaaD by bromide ion. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 December 15. Published in final edited form as:Biochemistry. The catabolism of 1,3-dichloropropene (1, Scheme 1) in Pseudomonas pavonaceae 170 and coryneform bacterium strain FG41 i...

Reactions of Cg10062, a cis-3-Chloroacrylic Acid Dehalogenase Homologue, with Acetylene and Allene Substrates: Evidence for a Hydration-Dependent Decarboxylation

“…The substantial increase in K m (~687-fold) observed for Cg10062 using ( cis -3-bromoacrylate) suggests that it might originate in the elements that govern substrate specificity. 11,12 Two possibilities include the more spacious active site of Cg10062 or a six-residue loop in cis -CaaD that is observed closed down on the active site in the crystal structure of the enzyme inactivated by an irreversible inhibitor. 11 A similar feature has not been observed in Cg10062.…”

Kinetic and structural characterization of a cis -3-Chloroacrylic acid dehalogenase homologue in Pseudomonas sp. UW4: A potential step between subgroups in the tautomerase superfamily

“…cis -CaaD and Cg10062 share 32% identity. 9,10,40 The Ps01740 trimer can be superimposed on that of cis -CaaD with a root-mean-square deviation (RMSD) of 1.14 Å (Cα) and on that of Cg10062 with a RMSD of 0.975Å (Cα) (Figure 2B). Finally, a structural superimposition of the monomers with the 4-OT dimer shows that the major structural differences are in the loops connecting the two β–α–β subunits and the C-termini of the monomers (Figure 2C).…”

“…9 The enzyme has been extensively characterized by kinetic, mechanistic, and structural studies. 1,2,10,11 It is a trimer where each monomer consists of 149 amino acids that code for two fused β–α–β motifs. Six active site amino acids (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114) have been implicated in the catalytic mechanism.…”

Resolution of the uncertainty in the kinetic mechanism for the trans -3-Chloroacrylic acid dehalogenase-catalyzed reaction

“…The dissociation constant for bromide is comparable (10 mM for CaaD and 3 mM for cis -CaaD), but CaaD binds malonate semialdehyde ( 4 ) more tightly (the K D is 10-fold less than that for bromide) than cis -CaaD (where the K D is 10-fold greater than that for bromide). 13 Both enzymes are limited by product release and not chemistry. In contrast, Horvat et al reported that product release is not rate limiting for CaaD, based on the observation that increasing the solvent viscosity did not decrease the values of k cat or k cat / K m .…”