Determination of the chemical pathway for 4-chlorobenzoate: Coenzyme a ligase catalysis

4-Chlorobenoate:coenzyme A ligase (4-CBA:CoA ligase) catalyzes the first step of the 4-CBA degradation pathway of Pseudomonas sp. strain CBS3. In this reaction, 4-CBA-CoA thioester synthesis is coupled to ATP cleavage. The studies described in this paper examine the intermediacy of 4-chlorobenzoyl-adenosine 5'-phosphate diester (4-CBA-AMP) in the ligase reaction. The I-CBA-AMP adduct was isolated from the ligase reaction mixture generated from magnesium adenosine 5-triphosphate (MgATP) and 4-CBA in the absence of CoA. The structure of the 4-CBA-AMP was verified by H-1-, C-13-, and P-31-nuclear magnetic resonance analysis. Single-turnover reactions carried out with C-14-labeled 4-CBA in a rapid quench apparatus demonstrated formation of the enzyme 4 . CBA-AMP . MgPPi complex from the enzyme . 4-CBA . MgATP complex at a rate of 135 s(-1). The rate of ligand release from the enzyme . 4-CBA-AMP . MgPPi complex was measured at 0.013 s(-1) Single-turnover reactions of [C-14]-4-CBA, MgATP, and CoA catalyzed by the ligase revealed that the 4-CBA-AMP intermediate formed reaches a maximum level of 25% of the starting 4-CBA within 10 ms and then declines with the formation of the 4-CBA-CoA. The rates of the adenylation and thioesterification partial reactions, determined by kinetic simulation of the rate data, are nearly equal (135 and 100 s(-1)). Substitution of CoA with the slow substrate pantetheine did not significantly alter the rate of the adenylation step but did reduce the rate of the thioesterification step to 2 s(-1). The maximum level of 4-CBA-AMP reached during the single-turnover reaction of 4-CBA, MgATP, and pantetheine corresponded to one-half of the starting 4-CBA.