Mechanistic differences among retaining disaccharide phosphorylases:: insights from kinetic analysis of active site mutants of sucrose phosphorylase and α,α-trehalose phosphorylase
Mechanistic differences among retaining disaccharide phosphorylases:: insights from kinetic analysis of active site mutants of sucrose phosphorylase and α,α-trehalose phosphorylase
复制标题
DOI：
10.1016/j.carres.2008.01.029
复制
发表时间：
2008-08-11
影响因子：
3.1
通讯作者：
Nidetzky, Bernd
中科院分区：
文献类型：
--
作者：
Goedl, Christiane;Schwarz, Alexandra;Nidetzky, Bernd
蔗糖磷酸化酶利用糖苷水解酶样双置换机制将其二糖底物和磷酸盐转化为α-D-葡萄糖1-磷酸和果糖。采用定点突变的方法研究了肠膜明串珠菌蔗糖磷酸化酶中Asp（196）和Glu（237）的催化亲核试剂和酸碱作用。表明Asp（295）的侧链通过与葡糖基氧碳正离子样物质的2-羟基的强氢键（>= 23 kJ/mol）促进Asp（196）的葡糖基化和去葡糖基化的催化步骤，所述葡糖基氧碳正离子样物质被认为在β-葡糖基酶中间体侧翼的过渡态中形成。用于检查裂褶菌α，α-海藻糖磷酸化酶的α-保留葡糖基转移的机制的生物化学技术的分类未能提供支持通过共价中间体的类似的两步催化反应的证据。诱变研究表明，这种海藻糖磷酸化酶的活性位点结构是典型的保留折叠家庭GT-B的糖基转移酶和蔗糖磷酸化酶显着不同。虽然关于海藻糖磷酸化酶功能的化学机制仍然不明确，但这两种二糖磷酸化酶已经进化出显著不同的反应坐标，以在其高度相似的底物转化中实现催化效率和立体化学控制。(C)2008爱思唯尔有限公司保留所有权利。
Sucrose phosphorylase utilizes a glycoside hydrolase-like double displacement mechanism to convert its disaccharide substrate and phosphate into alpha-D-glucose 1-phosphate and fructose. Site-directed mutagenesis was employed to characterize the proposed roles of Asp(196) and Glu(237) as catalytic nucleophile and acid-base, respectively, in the reaction of sucrose phosphorylase from Leuconostoc mesenteroides. The side chain of Asp(295) is suggested to facilitate the catalytic steps of glucosylation and deglucosylation of Asp(196) through a strong hydrogen bond (>= 23 kJ/mol) with the 2-hydroxyl of the glucosyl oxocarbenium ion-like species believed to be formed in the transition states flanking the beta-glucosyl enzyme intermediate. An assortment of biochemical techniques used to examine the mechanism of alpha-retaining glucosyl transfer by Schizophyllum commune alpha,alpha-trehalose phosphorylase failed to provide evidence in support of a similar two-step catalytic reaction via a covalent intermediate. Mutagenesis studies suggested a putative active-site structure for this trehalose phosphorylase that is typical of retaining glycosyltransferases of fold family GT-B and markedly different from that of sucrose phosphorylase. While ambiguity remains regarding the chemical mechanism by which the trehalose phosphorylase functions, the two disaccharide phosphorylases have evolved strikingly different reaction coordinates to achieve catalytic efficiency and stereochemical control in their highly analogous substrate transformations. (C) 2008 Elsevier Ltd. All rights reserved.