SPECIFICITY OF 4-CHLOROBENZOYL COENZYME-A DEHALOGENASE CATALYZED DEHALOGENATION OF HALOGENATED AROMATICS

Steady-state and transient kinetic techniques were used to evaluate th e efficiency of 4-chlorobenzoyl coenzyme A (4-CBA-CoA) turnover cataly zed by 4-CBA-CoA dehalogenase from Pseudomonas sp. CBS-3. The k(cat) f or a single turnover on the enzyme was found to be 2 s-1, while that f or multiple turnovers was found to be 0.6 s-1. Catalysis rather than p roduct release was judged to be rate limiting. Comparison of the rates of turnover of 4-bromobenzoyl-CoA (1.4 s-1), 4-iodobenzoyl-CoA (1.1 s -1), and 4-fluorobenzoyl-CoA (8 X 10(-6) s-1) indicated that cleavage of the carbon-halogen bond occurs in the rate-limiting transition stat e of the reaction. Structure-activity measurements made with 4-CBA-CoA analogs bearing electron-donating or -withdrawing substituents at C(2 ) or C(3) suggested the importance of steric/solvation effects on the enzymatic reaction and failed to provide insight into the nature of th e reaction intermediate. The inhibition constants measured for benzoyl -CoA (72 muM), CoA (140 muM), and 4-chlorobenzoate (21 mM) compared to the K(m) measured for 4-CBA-CoA (4 muM) suggest the dominant role pla yed by the CoA moiety in substrate anchoring.