EXPLORATION OF POSSIBLE MECHANISMS FOR 4-CHLOROBENZOYL COA DEHALOGENASE - EVIDENCE FOR AN ARYL-ENZYME INTERMEDIATE

4-Chlorobenzoyl CoA dehalogenase catalyzes the replacement of the chlo rine substituent on 4-chlorobenzoyl CoA with a hydroxyl group. The SNA r mechanism seems the most likely mechanism for this unusual and intri nsically difficult nucleophilic aromatic substitution reaction. Howeve r, the order of leaving group abilities observed for various 4-haloben zoyl CoA substrates is opposite that expected. Therefore, we have expl ored alternative mechanisms for the enzymic dehalogenation reaction. T he aryne mechanism was ruled out by the absence of a deuterium kinetic isotope effect on the reaction. The S(RN)1 and S(ON)2 mechanisms were deemed unlikely because of the lack of evidence for a metal ion or or ganic cofactor on the enzyme. Thus, the dehalogenation reaction appear s to occur via an SNAr mechanism, Further investigations suggested tha t the reaction proceeds by displacement of chloride by an enzymic carb oxylate, followed by hydrolysis of an aryl-enzyme intermediate. When a n alternative nucleophile, hydroxylamine, was included in reaction mix tures, no product derived from direct attack of hydroxylamine upon 4-c hlorobenzoyl CoA could be detected. However, inclusion of higher conce ntrations of hydroxylamine (100 mM) resulted in inactivation of the en zyme. These data are consistent with the formation of an aryl-enzyme i ntermediate that is converted to a hydroxamic acid upon attack by hydr oxylamine. Enzyme activity is recovered after hydroxylamine is removed , suggesting that the enzyme is able to slowly hydrolyze the hydroxami c acid and restore the active-site carboxylate, Single-turnover O-18-l abeling experiments designed to confirm that the reaction occurs by di rect attack of an active-site carboxylate to form an aryl-enzyme inter mediate were difficult to interpret. Approximately one-half of the pro duct contained oxygen derived from the solvent and one-half contained oxygen derived from the enzyme. Possible explanations for this phenome non were explored, but a satisfactory explanation has not been found.