Examining the role of phosphate in glycosyl transfer reactions of Cellulomonas uda cellobiose phosphorylase using d-glucal as donor substrate

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• 作者：Patricia Wildberger^a ; Lothar Brecker^b ; Bernd Nidetzky^a ; ^{bernd.nidetzky@tugraz.at}
• 关键词：Cellobiose phosphorylase ; Glycoside hydrolase family GH-94 ; d-Glucal ; Catalytic mechanism ; Stereospecific protonation ; 2-Deoxy-d-glucosyl transfer
• 刊名：Carbohydrate Research
• 出版年：2012
• 期刊代码：15_00086215
• 类别：ch
• 出版时间：15 July, 2012
• 卷：356
• 期：Complete
• 页码：224-232
• 文件大小：751 K

摘要

Cellobiose phosphorylase from Cellulomonas uda (CuCPase) is shown to utilize d-glucal as slow alternative donor substrate for stereospecific glycosyl transfer to inorganic phosphate, giving 2-deoxy-伪-d-glucose 1-phosphate as the product. When performed in D₂O, enzymatic phosphorolysis of d-glucal proceeds with incorporation of deuterium in equatorial position at C-2, implying a stereochemical course of reaction where substrate becomes protonated from below its six-membered ring through stereoselective re side attack at C-2. The proposed catalytic mechanism, which is supported by results of docking studies, involves direct protonation of d-glucal by the enzyme-bound phosphate, which then performs nucleophilic attack on the reactive C-1 of donor substrate. When offered d-glucose next to d-glucal and phosphate, CuCPase produces 2-deoxy-尾-d-glucosyl-(1鈫?)-d-glucose and 2-deoxy-伪-d-glucose 1-phosphate in a ratio governed by mass action of the two acceptor substrates present. Enzymatic synthesis of 2-deoxy-尾-d-glucosyl-(1鈫?)-d-glucose is effectively promoted by catalytic concentrations of phosphate, suggesting that catalytic reaction proceeds through a quaternary complex of CuCPase, d-glucal, phosphate, and d-glucose. Conversion of d-glucal and phosphate presents a convenient single-step synthesis of 2-deoxy-伪-d-glucose 1-phosphate that is difficult to prepare chemically.