Zhuang Zhihao, Song Feng, Zhang Wenhai, Taylor Kimberly, Archambault Angela, Dunaway-Mariano Debra, Dong Jian, Carey Paul R
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Biochemistry. 2002 Sep 17;41(37):11152-60. doi: 10.1021/bi0262303.
4-Hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase catalyzes the hydrolysis of 4-HBA-CoA to 4-hydroxybenzoate and CoA. X-ray crystallographic analysis of the liganded enzyme has shown that the benzoyl thioester and pantetheine moieties of the substrate ligand are bound in a narrow crevice while the nucleotide moiety rests on the protein surface (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., in press). Asp17 is positioned in the crevice, close to the substrate thioester C=O, which in turn interacts with the positive pole of an alpha-helix macrodipole. In this paper we report the results from spectral, mutagenesis, and kinetic studies which show (1) that substrate activation involves restricted thioester C=O conformational freedom and a modest enhancement of C=O bond polarization, (2) that the nucleotide unit of the substrate is bound through interaction with the protein surface, and (3) that Asp17 contributes a rate factor of 10(4), consistent with its proposed role of general base or nucleophile.
4-羟基苯甲酰辅酶A（4-HBA-CoA）硫酯酶催化4-HBA-CoA水解生成4-羟基苯甲酸和辅酶A。对结合配体的该酶进行的X射线晶体学分析表明，底物配体的苯甲酰硫酯和泛酰巯基乙胺部分结合在一个狭窄的裂隙中，而核苷酸部分则位于蛋白质表面（托登，J.B.，霍尔登，H.M.，庄，Z.和邓纳韦-马里亚诺，D.（2002年）野生型和突变型4-羟基苯甲酰辅酶A硫酯酶抑制剂与底物复合物的X射线晶体学分析，《生物化学杂志》，即将发表）。天冬氨酸17位于裂隙中，靠近底物硫酯的C=O，该C=O又与α-螺旋大偶极的正极相互作用。在本文中，我们报告了光谱、诱变和动力学研究的结果，这些结果表明：（1）底物激活涉及硫酯C=O构象自由度受限和C=O键极化适度增强；（2）底物的核苷酸单元通过与蛋白质表面的相互作用而结合；（3）天冬氨酸17贡献了10⁴的速率因子，与其作为通用碱或亲核试剂的假定作用一致。
