Catalytic mechanism of human UDP-glucose 6-dehydrogenase: in situ proton NMR studies reveal that the C-5 hydrogen of UDP-glucose is not exchanged with bulk water during the enzymatic reaction

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• 作者：Thomas Eixelsberger^a ; Lothar Brecker^b ; Bernd Nidetzky^a ; ^{bernd.nidetzky@tugraz.at}
• 关键词：UDP-glucose dehydrogenase ; Thiohemiacetal and thioester intermediates ; Catalytic mechanism ; Deuterium incorporation ; UDP-gluco-hexodialdose
• 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：209-214
• 文件大小：815 K

摘要

Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes the biosynthetic oxidation of UDP-glucose into UDP-glucuronic acid. The catalytic reaction proceeds in two NAD⁺-dependent steps via covalent thiohemiacetal and thioester enzyme intermediates. Formation of the thiohemiacetal adduct occurs through attack of Cys²⁷⁶ on C-6 of the UDP-gluco-hexodialdose produced in the first oxidation step. Because previous studies of the related enzyme from bovine liver had suggested loss of the C-5 hydrogen from UDP-gluco-hexodialdose due to keto-enol tautomerism, we examined incorporation of solvent deuterium into product(s) of UDP-glucose oxidation by hUGDH. We used wild-type enzyme and a slow-reacting Glu¹⁶¹鈫扜ln mutant that accumulates the thioester adduct at steady state. In situ proton NMR measurements showed that UDP-glucuronic acid was the sole detectable product of both enzymatic transformations. The product contained no deuterium at C-5 within the detection limit (猢?%). The results are consistent with the proposed mechanistic idea for hUGDH that incipient UDP-gluco-hexodialdose is immediately trapped by thiohemiacetal adduct formation.