Identification of Tyr241 as a Key Catalytic Base in the Family 4 Glycoside Hydrolase BgIT from Thermotoga maritima
Identification of Tyr241 as a Key Catalytic Base in the Family 4 Glycoside Hydrolase BgIT from Thermotoga maritima
复制标题
DOI：
10.1021/bi301021u
复制
发表时间：
2012-10-23
期刊：
影响因子：
2.9
通讯作者：
Withers, Stephen G.
中科院分区：
文献类型：
--
作者：
Yip, Vivian L. Y.;Withers, Stephen G.
虽然绝大多数糖苷酶通过类氧碳正离子的过渡态催化糖苷水解，通常使用羧酸残基作为酸/碱或亲核催化剂，但这些酶的两个亚家族(CAZY分类中的GH4和GH109)通过涉及阴离子过渡态的氧化还原辅助机制进行水解。虽然已经获得了关于这一机制的良好证据，但所涉及的催化残留物的身份尚未得到确认。对来自Thermotoga maritima(BglT)的6-磷酸-β-葡萄糖苷酶(BglT)突变的机理分析，其中活性位点酪氨酸残基(Tyr 241)已被Phe和Ala取代，为其作为催化碱基的作用提供了支持。相对于野生型酶，k(CAT)和k(CAT)/K-m的pH依赖关系，尤其是与碱基对应的酸性支链的依赖关系发生了漂移。Tyr241突变体对在C1、C2和C3处氢化的底物的动力学同位素效应强烈依赖于pH，在低pH下与Tyr241Ala突变体一起观察到2-氚底物的基本完全的一级动力学同位素效应。这与移除碱基后去质子化步骤的减慢是一致的。
While the vast majority of glycosidases catalyze glycoside hydrolysis via oxocarbenium ion like transition states and typically employ carboxylic acid residues as acid/base or nucleophile catalysts, two subfamilies of these enzymes (GH4 and GH109 in the CAZY Classification) conduct hydrolysis via a redox-assisted mechanism involving anionic transition states. While good evidence of this mechanism has been obtained, the identities of the catalytic residues involved have not yet been confirmed. Mechanistic analyses of mutants of the 6-phospho-beta-glucosidase from Thermotoga maritima (BglT), in which the active site tyrosine residue (Tyr 241) has been replaced with Phe and Ala, provide support for its role as a catalytic base. The pH dependence of k(cat), and k(cat)/K-m, particularly of the acidic limb corresponding to the base, is shifted relative to that of the wild type enzyme. Kinetic isotope effects for hydrolysis of substrates deuterated at C1, C2, and C3 by the Tyr 241 mutants are strongly pH-dependent, with essentially full primary kinetic isotope effects being observed for the 2-deutero substrate at low pH with the Tyr241Ala mutant. This is consistent with a slowing of the deprotonation step upon removal of the base.