Alonso M D, Lomako J, Lomako W M, Whelan W J, Preiss J
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, FL 33101.
FEBS Lett. 1994 Sep 26;352(2):222-6. doi: 10.1016/0014-5793(94)00962-7.
Glycogenin, the self-glucosylating primer for glycogen synthesis, is expressed in wild-type E. coli as a recombinant protein in an already partly glucosylated form, owing to the presence of its substrate, UDP-glucose. By using an E. coli mutant strain lacking in UDP-glucose pyrophosphorylase activity, we have succeeded in expressing carbohydrate-free glycogenin (apo-glycogenin) in good yield. When provided with UDPxylose, it autocatalytically adds 1 xylose residue. With UDP-glucose, an average of 8 glucose residues are added. However, release of the self-synthesized maltosaccharide chains with isoamylase reveals them to be a mixture. Chains as long as 11 glucose residues (maltoundecaose) are present. The ability of recombinant apo-glycogenin to self-glucosylate is further proof that a separate enzyme is not needed for the addition of the first glucose residue to Tyr-194 of the protein.
糖原素是糖原合成的自糖基化引物，由于其底物尿苷二磷酸葡萄糖（UDP-葡萄糖）的存在，它在野生型大肠杆菌中以部分糖基化的重组蛋白形式表达。通过使用缺乏UDP-葡萄糖焦磷酸化酶活性的大肠杆菌突变菌株，我们成功地以高产率表达了无碳水化合物的糖原素（脱辅基糖原素）。当提供UDP-木糖时，它会自动催化添加1个木糖残基。使用UDP-葡萄糖时，平均会添加8个葡萄糖残基。然而，用异淀粉酶释放自合成的麦芽糖链后发现它们是混合物。存在长达11个葡萄糖残基的链（麦芽十一糖）。重组脱辅基糖原素的自糖基化能力进一步证明，在蛋白质的酪氨酸-194上添加第一个葡萄糖残基不需要单独的酶。
