Exploration of possible mechanisms for 4-chlorobenzoyl CoA dehalogenase: Evidence for an aryl-enzyme intermediate

4-Chlorobenzoyl CoA dehalogenase catalyzes the replacement of the chlorine substituent on 4-chlorobenzoyl CoA with a hydroxyl group. The S sub(N)Ar mechanism seems the most likely mechanism for this unusual and intrinsically difficult nucleophilic aromatic substitution reaction. However, the order of leaving group abilities observed for various 4-halobenzoyl CoA substrates is opposite that expected. Therefore, we have explored alternative mechanisms for the enzymic dehalogenation reaction. The aryne mechanism was ruled out by the absence of a deuterium kinetic isotope effect on the reaction. The S sub(RN)1 and S sub(ON)2 mechanisms were deemed unlikely because of the lack of evidence for a metal ion or organic cofactor on the enzyme. Thus, the dehalogenation reaction appears to occur via an S sub(N)Ar mechanism. Further investigations suggested that the reaction proceeds by displacement of chloride by an enzymic carboxylate, followed by hydrolysis of an aryl-enzyme intermediate. When an alternative nucleophile, hydroxylamine, was included in reaction mixtures, no product derived from direct attack of hydroxylamine upon 4-chlorobenzoyl CoA could be detected. However, inclusion of higher concentrations of hydroxylamine (100 mM) resulted in inactivation of the enzyme. These data are consistent with the formation of an aryl-enzyme intermediate that is converted to a hydroxamic acid upon attack by hydroxylamine. Enzyme activity is recovered after hydroxylamine is removed, suggesting that the enzyme is able to slowly hydrolyze the hydroxamic acid and restore the active-site carboxylate. Single-turnover super(18)O-labeling experiments designed to confirm that the reaction occurs by direct attack of an active-site carboxylate to form an aryl-enzyme intermediate were difficult to interpret. Approximately one-half of the product contained oxygen derived from the solvent and one-half contained oxygen derived from the enzyme. Possible explanations for this phenomenon were explored, but a satisfactory explanation has not been found. Several soil microorganisms, including Acinetobacter sp. 4-CB1, Pseudomonas CBS-3, coryneform bacterium NTB-1, and various strains of Arthrobacter sp. have been reported to degrade 4-chlorobenzoate, a breakdown product of some PCB's.