A Pre-Steady State Kinetic Analysis of the αY60W Mutant oftrans-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-chloracrylic 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 carries out 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.

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

“…CaaD lacks a strongly fluorescent amino acid in or near the active site, so one was introduced. 12 The resulting αY60W mutant of CaaD shows little kinetic differences from those of the wild-type, and accurately reports changes in enzyme fluorescence during the CaaD-conversion of trans -3-bromoacrylate ( 9 , Scheme 3) to 4 , in both the transient state and steady-state realms.…”

“…12–14 The DEAE-Sepharose and Phenyl-Sepharose 6 Fast Flow resins were obtained from GE Healthcare Bio-Sciences (Pittsburgh, PA). The EMD Millipore ultrafiltration membranes (10,000 Da MW cutoff) were purchased from Fisher Scientific.…”

“…The construction of the αY60W-CaaD plasmid, transformation into E. coli BL21-Gold (DE3) cells, and expression are described elsewhere. 12 Cells were grown at 37 °C in Luria-Bertani (LB) media that contained kanamycin (Kn, 30 μg/mL). The αY60W mutant of CaaD was purified as reported.…”

“…12 The fit established a minimal kinetic model for the CaaD reaction with estimates on six individual rate constants (Scheme 4), including chemistry ( k 2 ), product release ( k 4 ) and binding constants for the substrate ( K D = k −1 / k 1 ) and products ( 4 and bromide, K 5 and K 6 , respectively). Limits (lower and upper, respectively) were placed on k 1 and k −3 , but their absolute values were not defined by the data.…”

“…12 First, the data set did not define the order of product release ( P , in Scheme 4) from the E * PQ species. Binding of P to E ** Q could only be assessed as weak, with an estimated K D > 200 mM.…”