Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, New Mexico 87131, United States.
Biochemistry. 2012 Jan 24;51(3):786-94. doi: 10.1021/bi2013917. Epub 2012 Jan 13.
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, 27468-27476]. 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 μM [(14)C]-4-HB-CoA catalyzed by 10 μM 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 μM; K(ii) = 900 ± 300 μM) and 4-HB (K(is) = 1.2 ± 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D(2)O 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 μM 4-HB-CoA and 0.54 μM thioesterase), single-turnover reaction (5 μM [(14)C]-4-HB-CoA catalyzed by 50 μM thioesterase), steady-state reaction (5.2 μM 4-HB-CoA catalyzed by 0.003 μM thioesterase), and transient-state multiple-turnover reaction (50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase). Together with the results obtained from solvent (18)O 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.
来自假单胞菌 CBS3 菌株的 4-羟基苯甲酰辅酶 A (4-HB-CoA)硫酯酶催化 4-氯苯甲酸降解途径的最后一步，即 4-HB-CoA 水解为辅酶 A (CoA)和 4-羟基苯甲酸 (4-HB)。在以前的工作中，底物结合硫酯酶的 X 射线结构分析提供了活性位点天冬氨酸残基 17 在亲核催化中的作用的证据[Thoden，J. B.，Holden，H. M.，Zhuang，Z.，和 Dunaway-Mariano，D. (2002) X 射线晶体学分析野生型和突变体 4-羟基苯甲酰辅酶 A 硫酯酶的抑制剂和底物复合物。J. Biol. Chem. 277, 27468-27476]。在本研究中，使用动力学技术测试了 X 射线结构数据所暗示的催化机制。用 10 μM 硫酯酶催化 50 μM [(14)C]-4-HB-CoA 的多次转化反应的时间过程支持两步途径，其中第二步是限速步骤。稳态产物抑制研究表明，CoA (K(is) = 250 ± 70 μM；K(ii) = 900 ± 300 μM)和 4-HB (K(is) = 1.2 ± 0.2 mM)的结合较弱，表明产物释放不是限速步骤。稳态 k(cat) 值(18 s(-1))的 D(2)O 溶剂动力学同位素效应(3.8)很大，这表明涉及质子转移的化学步骤是限速步骤。总的来说，动力学结果支持双化学途径。通过基于模拟的拟合确定控制假定的天冬氨酸 17-(4-羟基苯甲酰)酰化物中间物形成和消耗的微观速率常数，该拟合基于底物结合反应(5.0 μM 4-HB-CoA 和 0.54 μM 硫酯酶)、单轮反应(5 μM [(14)C]-4-HB-CoA 由 50 μM 硫酯酶催化)、稳态反应(0.003 μM 硫酯酶催化 5.2 μM 4-HB-CoA)和瞬态多次转化反应(10 μM 硫酯酶催化 50 μM [(14)C]-4-HB-CoA)的时间过程。与从溶剂(18)O 标记实验获得的结果一起，这些发现被解释为形成天冬氨酸 17-(4-羟基苯甲酰)酰化物中间物的证据，该中间物在羟基苯甲酰羰基碳原子上经历限速水解裂解。
