A persistent pesticide residue and the unusual catalytic proficiency of a dehalogenating enzyme

Abstract

The soil of potato fields in The Netherlands harbors bacteria with the ability to metabolize 3-chloroacrylic acid, generated by the degradation of a pesticide (1,3-dichloropropene) that entered the environment in 1946. From examination of rate constants at elevated temperatures, we infer that the half-time at 25 degrees C for spontaneous hydrolytic dechlorination of trans-3-chloroacrylic acid is 10,000 years, several orders of magnitude longer than half-times for spontaneous decomposition of other environmental pollutants such as 1,2-dichloroethane (72 years), paraoxon (13 months), atrazine (5 months), and aziridine (52 h). With thermodynamic parameters for activation similar to those for the spontaneous hydration of fumarate at pH 6.8, this slow reaction proceeds at a constant rate through the pH range between 2 and 12. However, at the active site of the enzyme 3-chloroacrylate dehalogenase (CaaD), isolated from a pseudomonad growing in these soils, hydrolytic dechlorination proceeds with a half-time of 0.18 s. Neither k(cat) nor k(cat)/K(m) is reduced by increasing solvent viscosity with trehalose, implying that the rate of enzymatic dechlorination is controlled by chemical events in catalysis rather than by diffusion-limited substrate binding or product release. CaaD achieves an approximately 10(12)-fold rate enhancement, matching or surpassing the rate enhancements produced by many enzymes that act on more conventional biological substrates. One of those enzymes is 4-oxalocrotonate tautomerase, with which CaaD seems to share a common evolutionary origin.

Fig. 1.

Reaction catalyzed by CaaD and fumarase.

Fig. 2.

Effect of temperature on the first-order rate constant for dehalogenation of trans-3-chloroacrylic acid (0.01 M) in potassium phosphate buffer (0.1 M, pH 8.0).

Fig. 3.

Rate constants for the uncatalyzed decomposition at 25°C of 3-chloroacrylate (this work), 1,2-dichloroethane (18), chloroethane (19), 1,2-dibromoethane (19), paraoxon (20), atrazine (21), 1,3-dichloropropene (22), and aziridine (23).

Fig. 4.

Rate constants, equilibrium constants, and thermodynamics of activation for the CaaD-catalyzed and uncatalyzed dehalogenation of trans-3-chloroacrylic acid at 25°C, determined in potassium phosphate buffer (0.02 M, pH 8.0).

Fig. 5.

Rate constants for some enzyme-catalyzed and uncatalyzed reactions at 25°C, including orotidylate decarboxylase (ODC), β-amylase (GLU), fumarase (FUM) (13), mandelate racemase (MAN), CaaD (this work), urease (URE), cytidine deaminase (CDA), ketosteroid isomerase (KSI), chorismate mutase (CMU), 4-OT (18), and carbonic anhydrase (CAN). For references, see Table 1.

Fig. 6.

Influence of relative viscosity in the presence of added trehalose on k_cat (A) and k_cat/K_m (B) for CaaD in potassium phosphate buffer (0.02 M, pH 8.0) at 25°C.