Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, NM 87131, USA.
Biochemistry. 2012 Sep 4;51(35):7000-16. doi: 10.1021/bi301059m. Epub 2012 Aug 20.
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 (18)O-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 (31)P 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.
来自节杆菌属 AU 菌株的热狗折叠酶 4-羟基苯甲酰辅酶 A（4-HB-CoA）硫酯酶在 4-氯苯甲酸脱卤途径的最后一步催化 4-HB-CoA 水解生成 4-羟基苯甲酸（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.，Zhuang，Z.，Dunaway-Mariano，D.，和 Holden H. M.（2003）J. Biol. Chem. 278，43709-43716），设计了一系列定点突变体来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天然折叠，在某些情况下，揭示活性位点构象的变化。同时，野生型和突变硫酯酶进行瞬态和稳态动力学分析以及（18）O-溶剂标记实验。提供的证据表明，Glu73 在亲核催化中起作用，Gly65 和 Gln58 通过与硫酯部分形成氢键来促进过渡态稳定，而 Thr77 或定向水分子亲核试剂攻击酶-酰基中间物的 4-羟基苯甲酰碳。用 Asp 替换 Glu73 表明羧酸盐残基的功能从亲核催化转变为碱催化，因此，反应从涉及共价酶中间物的两步过程转变为单步水解反应。E73D/T77A 双突变体恢复了 E73D 单突变体丢失的大部分催化效率。31P NMR 实验的结果表明，底物核苷酸单元与酶表面结合。对定点突变体的动力学分析表明 Arg102、Arg150、Ser120 和 Thr121 对结合核苷酸单元有贡献。最后，我们通过对 Asp31、His64 和 Glu78 定点突变体的动力学和 X 射线分析表明，这三个活性位点残基对底物 4-羟基苯甲酰环的有效结合很重要。
