Steady-state and transient kinetic techniques were used to evaluate the efficiency of 4-chlorobenzoyl coenzyme A (4-CBA-CoA) turnover catalyzed by 4-CBA-CoA dehalogenase from Pseudomonas sp. CBS-3. The kcat for a single turnover on the enzyme was found to be 2 s-1, while that for multiple turnovers was found to be 0.6 s-1. Catalysis rather than product 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 state 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 the reaction intermediate. The inhibition constants measured for benzoyl-CoA (72 microM), CoA (140 microM), and 4-chlorobenzoate (21 mM) compared to the Km measured for 4-CBA-CoA (4 microM) suggest the dominant role played by the CoA moiety in substrate anchoring.