The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp Strain SU
The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp Strain SU
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DOI：
10.1021/bi301059m
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发表时间：
2012-09-04
期刊：
影响因子：
2.9
通讯作者：
Dunaway-Mariano, Debra
中科院分区：
文献类型：
--
作者：
Song, Feng;Thoden, James B.;Dunaway-Mariano, Debra
来自节杆菌属菌株Au的热狗折叠酶4-羟基苯甲酰基-辅酶A（4-HB-CoA）硫酯酶在4-氯苯甲酸脱卤途径的最后步骤中催化4-HB-CoA水解形成4-羟基苯甲酸酯（4-HB）和辅酶A（CoA）。根据已发表的配体酶的X射线结构（Thoden，J. B.，庄志，字：Dunaway-Mariano，D.，和霍尔顿H. M.（2003）J.Biol.Chem.278，43709-43716），制备了一系列定点突变体用于测试活性位点残基在底物结合和催化中的作用。突变硫酯酶进行X-射线结构测定，以确认保留的天然折叠，并在某些情况下，以揭示活性位点构型的变化。同时，野生型和突变硫酯酶进行瞬态和稳态动力学分析，并O-18-溶剂标记实验。提供的证据表明，Glu 73的功能在亲核催化，Gly 65和Gln 58有助于通过与硫酯部分形成氢键的过渡态稳定化和Thr 77定向的水亲核试剂的攻击在4-羟基苯甲酰基碳的酶-酸酐中间体。用Asp取代Glu 73显示出将羧酸残基的功能从亲核催化切换到碱催化，因此，反应从涉及共价酶中间体的两步过程切换到单步水解反应。E73 D/T77 A双突变体恢复了E73 D单突变体中损失的大部分催化效率。从P-31 NMR实验的结果表明，底物核苷酸单元结合到酶表面。进行定点突变体的动力学分析以确定Arg 102、Arg 150、Ser 120和Thr 121在结合核苷酸单元中所作的贡献。最后，我们表明Asp 31，His 64和Glu 78定点突变体的动力学和X-射线分析，这三个活性位点残基是重要的生产性结合的底物4-羟基苯甲酰基环。
The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp. strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden H. M. (2003) J. Biol. Chem. 278, 43709-43716), a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to O-18-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from P-31 NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.