Investigation of the Catalytic Mechanism of the Hotdog-Fold Enzyme Superfamily Pseudomonas sp. Strain CBS3 4-Hydroxybenzoyl-CoA Thioesterase

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• 作者：Zhihao Zhuang ; John Latham ; Feng Song ; Wenhai Zhang ; Michael Trujillo ; Debra Dunaway-Mariano
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：January 24, 2012
• 年：2012
• 卷：51
• 期：3
• 页码：786-794
• 全文大小：442K
• 年卷期：v.51,no.3(January 24, 2012)
• ISSN：1520-4995

文摘

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 Asp17 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鈥?7476]. 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 渭M [¹⁴C]-4-HB-CoA catalyzed by 10 渭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 卤 70 渭M; K_ii = 900 卤 300 渭M) and 4-HB (K_is = 1.2 卤 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^鈥?) 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 渭M 4-HB-CoA and 0.54 渭M thioesterase), single-turnover reaction (5 渭M [¹⁴C]-4-HB-CoA catalyzed by 50 渭M thioesterase), steady-state reaction (5.2 渭M 4-HB-CoA catalyzed by 0.003 渭M thioesterase), and transient-state multiple-turnover reaction (50 渭M [¹⁴C]-4-HB-CoA catalyzed by 10 渭M thioesterase). Together with the results obtained from solvent ¹⁸O 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.