Determination of the chemical pathway for 4-chlorobenzoate:coenzyme A ligase catalysis.
4-Chlorobenzoate: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 4-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 1H- 13C-, and 31P-nuclear magnetic resonance analysis. Single-turnover reactions carried out with 14C-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 [14C]-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.
Chang
K H
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Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.
Dunaway-Mariano
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D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D002723:Chlorobenzoates / Q000737:chemistry / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D010204:Pantetheine / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology
D002723:Chlorobenzoates; D011756:Diphosphates; D000249:Adenosine Monophosphate; D010204:Pantetheine; D000255:Adenosine Triphosphate; C077846:4-halobenzoate-CoA ligase; D003066:Coenzyme A Ligases; C040768:4-chlorobenzoic acid