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[摘要]：The 4-hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Pseudomonas sp. strain CBS3 catalyzes the final step of the 4-chlorobenzoate degradation pathway, which is the hydrolysis of 4-HB-CoA to coenzyme A (CoA) and 4-hydroxybenzoate (4-HB). In previous work, X-ray structural analysis of the substrate-bound thioesterase provided evidence of the role of an active site Asp 17 in nucleophilic catalysis [Thoden, J. B., Holden, H. M., Zhuang Z., and Dunaway-Mariano, D. (2002) X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase. J. Biol. Chem. 277, 27468-27476]. In the study presented here, kinetic techniques were used to test the catalytic mechanism that was suggested by the X-ray structural data. The time course for the multiple-turnover reaction of 50 mu M [C-14]-4-HB-CoA catalyzed by 10 mu M thioesterase supported a two-step pathway in which the second step is rate-limiting. Steady-state product inhibition studies revealed that binding of CoA (K-is = 250 +/- 70 mu M; K-ii = 900 +/- 300 mu M) and 4-HB (K-is = 1.2 +/- 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D2O solvent kinetic isotope effect (3.8) on the steady-state k(cat) value (18 s(-1)) provided evidence that a chemical step involving proton transfer is the rate-limiting step. Taken together, the kinetic results support a two-chemical pathway. The microscopic rate constants governing the formation and consumption of the putative aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate were determined by simulation-based fitting of a kinetic model to time courses for the substrate binding reaction (5.0 mu M 4-HB-CoA and 0.54 mu M thioesterase), single-turnover reaction (5 mu M [C-14] -4-HB-CoA catalyzed by 50 mu M thioesterase), steady-state reaction (5.2 mu M 4-HB-CoA catalyzed by 0.003 mu M thioesterase), and transient-state multiple-turnover reaction (50 mu M [C-14]-4-HB-CoA catalyzed by 10 mu M thioesterase). Together with the results obtained from solvent O-18 labeling experiments, the findings are interpreted as evidence of the formation of an aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate that undergoes rate-limiting hydrolytic cleavage at the hydroxybenzoyl carbonyl carbon atom. |
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