Chang S, Duerr B, Serif G
Department of Biochemistry, Ohio State University, Columbus 43210-1292.
J Biol Chem. 1988 Feb 5;263(4):1693-7.
The first committed enzyme in GDP-L-fucose formation from GDP-D-mannose is GDP-D-mannose 4,6-dehydratase, which forms GDP-4-keto-6-deoxy-D-mannose. The uncertain enzymatic steps beyond this point were examined in this study. Assays were developed for the epimerase and reductase activities which the putative pathway would predict. A protein was isolated exhibiting homogeneity by several criteria. This single protein, which forms GDP-L-fucose from GDP-4-keto-6-deoxy-D-mannose and NADH, appears to possess both epimerase and reductase capabilities and may be termed GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase. Analysis on a molecular sieve column using fast protein liquid chromatography established a molecular weight of 63,100 for the native enzyme, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis established a subunit molecular weight of 31,500.
从GDP-D-甘露糖生成GDP-L-岩藻糖过程中的第一个关键酶是GDP-D-甘露糖4,6-脱水酶，它能生成GDP-4-酮基-6-脱氧-D-甘露糖。本研究对这一步骤之后不确定的酶促反应步骤进行了研究。针对假定途径所预测的差向异构酶和还原酶活性开发了检测方法。通过多种标准分离出一种表现出均一性的蛋白质。这种能从GDP-4-酮基-6-脱氧-D-甘露糖和NADH生成GDP-L-岩藻糖的单一蛋白质似乎同时具备差向异构酶和还原酶能力，可被称为GDP-4-酮基-6-脱氧-D-甘露糖-3,5-差向异构酶-4-还原酶。使用快速蛋白质液相色谱在分子筛柱上进行分析，确定天然酶的分子量为63,100，而十二烷基硫酸钠-聚丙烯酰胺凝胶电泳确定亚基分子量为31,500。
