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 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.

Figure 1

Representative ion chromatograph of a quenched reaction mixture from a rapid quench experiment using cis-CaaD and 9 in 20 mM NaHCO₃ buffer, pH 9.0. The labeled peaks S and P correspond to 9 and bromide ion, respectively. The unlabeled peaks are unidentified buffer components. The eluting conditions are described in the text.

Figure 2

The cis-CaaD transient kinetic data fit globally to the single mechanism shown in Scheme 4 (solid line). (A) Stopped-flow fluorescence traces at various substrate concentrations (125–10,500 μM). For clarity, not all the traces are shown. (B) Rapid quench results for two concentrations of cis-CaaD (125 and 250 μM)³ and a saturating substrate concentration. In both Figures, the smooth lines were computed by numerical integration based upon the best global fit using Scheme 4 and the rate constants in Table 3.

Figure 3

The unliganded and covalently modified-cis-CaaD structures highlighting the loop region, consisting of residues 32-38 (11). (A) The active site cavity of cis-CaaD after incubation with (R)-oxirane-2-carboxylate (green). The Figure shows the prolyl nitrogen of Pro-1 attached to (R)-2-hydroxypropanoate (P1*). The carboxylate group of the adduct interacts with the backbone amide hydrogen atoms of Phe-37 and Leu-38 of the loop (shown in orange). Glu-114, Arg-70, Arg-73, and His-28 are also shown clockwise. (B) The active site cavity of the unliganded cis-CaaD structure (blue). The loop (shown in orange) points away from the active site cavity containing Glu-114, Glu-114, Arg-70, Arg-73, Pro-1, and His-28. This Figure was prepared with PyMOL (33).

Scheme 1

Scheme 2

Scheme 3

Scheme 4