L-fucose, 6-deoxy-L-galactose, is a key component of many important glycoconjugates including the blood group antigens and the Lewis(X) ligands. The biosynthesis of GDP-L-fucose begins with the action of a dehydratase that converts GDP-D-mannose into GDP-4-keto-6-deoxy-mannose. The enzyme GDP-fucose synthase, GFS, (also known as GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, GMER) then converts GDP-4-keto-6-deoxy-D-mannose into GDP-L-fucose. The GFS reaction involves epimerizations at both C-3'' and C-5'' followed by an NADPH-dependent reduction of the carbonyl at C-4. This manuscript describes studies that elucidate the order of the epimerization steps and the roles of the active site acid/base residues responsible for the epimerizations. An active site mutant, Cys109Ser, produces GDP-6-deoxy-D-altrose as its major product indicating that C-3'' epimerization occurs first and premature reduction of the GDP-4-keto-6-deoxy-D-altrose intermediate becomes competitive with GDP-L-fucose production. The same mutation results in the appearance of a kinetic isotope effect when [3''-(2)H]-GDP-6-deoxy-4-keto-mannose is used as a substrate. This indicates that Cys109 is the base responsible for the deprotonation of the substrate at C-3''. The Cys109Ser mutant also catalyzes a rapid wash-in of solvent derived deuterium into the C-5'' position of GDP-fucose in the presence of NADP(+). This confirms the order of epimerizations and the role of Cys109. Finally, the inactive His179Gln mutant readily catalyzes the wash-out of deuterium from the C-3'' position of [3''-(2)H]-GDP-6-deoxy-4-keto-mannose. Together these results strongly implicate an ordered sequence of epimerizations (C-3'' followed by C-5'') and suggest that Cys109 acts as a base and His179 acts as an acid in both epimerization steps.

中文翻译：

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GDP-岩藻糖合成酶的作用机制和活性位点残留

L-岩藻糖，6-脱氧-L-半乳糖，是包括血型抗原和Lewis(X) 配体在内的许多重要糖缀合物的关键成分。GDP-L-岩藻糖的生物合成开始于将GDP-D-甘露糖转化为GDP-4-酮-6-脱氧-甘露糖的脱水酶的作用。GDP-岩藻糖合酶 GFS（也称为 GDP-4-keto-6-deoxy-D-甘露糖差向异构酶/还原酶 GMER）然后将 GDP-4-keto-6-deoxy-D-甘露糖转化为 GDP- L-岩藻糖。GFS 反应涉及 C-3'' 和 C-5'' 的差向异构化，然后是 NADPH 依赖的 C-4 羰基还原。这份手稿描述了阐明差向异构化步骤顺序和负责差向异构化的活性位点酸/碱残基的作用的研究。活性位点突变体 Cys109Ser，生产 GDP-6-deoxy-D-altrose 作为其主要产品，表明 C-3'' 差向异构化首先发生，GDP-4-keto-6-deoxy-D-altrose 中间体的过早还原变得与 GDP-L-竞争岩藻糖生产。当 [3''-(2)H]-GDP-6-deoxy-4-keto-mannose 用作底物时，相同的突变会导致动力学同位素效应的出现。这表明 Cys109 是负责底物 C-3'' 去质子化的碱基。Cys109Ser 突变体还催化溶剂衍生的氘在 NADP(+) 存在下快速冲洗到 GDP-岩藻糖的 C-5'' 位置。这证实了差向异构化的顺序和 Cys109 的作用。最后，失活的 His179Gln 突变体很容易催化从 [3'' 的 C-3'' 位置洗出氘 -(2)H]-GDP-6-deoxy-4-keto-mannose。这些结果一起强烈暗示差向异构化的有序序列（C-3'' 后跟 C-5''），并表明 Cys109 在两个差向异构化步骤中充当碱，His179 充当酸。