EVIDENCE FOR NUCLEOPHILIC CATALYSIS IN THE AROMATIC-SUBSTITUTION REACTION CATALYZED BY (4-CHLOROBENZOYL)COENZYME-A DEHALOGENASE

(4-Chlorobenzoyl)coenzyme A dehalogenase catalyzes the hydrolytic dehalogenation of (4-chlorobenzoyl)coenzyme A (4-CBA-CoA) to (4-hydroxybenzoyl)coenzyme A (4-HBA-CoA). Rapid-quench techniques were used in conjunction with [C-14]-4-CBA-CoA to test for the formation of a covalent enzyme intermediate during catalysis. The rate of [C-14]-4-CBA-CoA (37 mu M) consumption in the presence of a 2-fold excess of dehalogenase (75 mu M) was determined to proceed at k = 6.5 s(-1), coincident with the formation of an enzyme intermediate containing covalently bound radiolabel. The radiolabeled enzyme reached a maximum level at 100 ms, corresponding to 27% of the starting [C-14]-4-CBA-CoA, before declining. The kinetics of formation and consumption of the radiolabeled enzyme observed during turnover are consistent with its intermediacy in the overall reaction. A single turnover reaction carried out in 98% O-18-enriched water produced 4-HBA-CoA with 73-75% O-16 and 27-25% O-18 at the benzoyl ring C(4)-OH. In contrast, a multiple turnover reaction carried out in 93% (H2O)-O-18 produced 4-HBA-CoA labeled at the C(4)-OH with 89% O-18 and 11% O-16. These results were interpreted as evidence for formation of an aryl enzyme intermediate during 4-CBA-CoA hydrolytic dechlorination in the dehalogenase active site.