Mutations of muscle glycogen synthase that disable activation by glucose 6-phosphate
Mutations of muscle glycogen synthase that disable activation by glucose 6-phosphate
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DOI：
10.1006/abbi.2001.2623
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发表时间：
2002-01-15
影响因子：
3.9
通讯作者：
Roach, PJ
中科院分区：
文献类型：
--
作者：
Hanashiro, I;Roach, PJ
糖原合酶是一种在可逆蛋白质修饰领域具有历史重要性的酶，其通过磷酸化失活并通过葡萄糖-6-磷酸（葡萄糖-6-P）变构活化。先前对酵母糖原合酶的分析已经鉴定了一个保守的高碱性13氨基酸片段，其中Arg残基的突变导致葡萄糖-6-P活化的丧失（12）。将等同突变R578 R579 R581 A（所有三个指示的Arg残基突变为Ala）和R585 R587 R590 A引入兔肌糖原合酶中。无论是在COS-1细胞中瞬时表达或在大肠杆菌中产生并纯化，这两种突变酶对葡萄糖-6-P的激活不敏感。磷酸化的影响以两种方式进行了研究。纯化的重组糖原合酶直接磷酸化酪蛋白激酶2和糖原合酶激酶3，在条件下，抑制野生型酶。此外，在野生型和COS-1细胞中表达的葡萄糖-6-P脱敏突变体中，通过诱变将磷酸化位点转化为Ala。磷酸化使R578 R579 R581 A突变体失活，但对R585 R587 R590 A几乎没有影响。该结果是令人惊讶的，因为磷酸化对相应的酵母酶突变体具有相反的作用。结果证实，该地区的糖原合成酶，精氨酸-578-精氨酸-590，是所需的葡萄糖-6-P的激活，并建议它是一个敏感的和关键的开关参与不同的构象状态之间的转换的一部分。然而，哺乳动物和酵母的酶之间的作用必须有微妙的不同。(C)2001年，爱思唯尔科学。
Glycogen synthase, an enzyme of historical importance in the field of reversible protein modification, is inactivated by phosphorylation and allosterically activated by glucose 6-phosphate (glucose-6-P). Previous analysis of yeast glycogen synthase had identified a conserved and highly basic 13-amino-acid segment in which mutation of Arg residues resulted in loss of activation by glucose-6-P (12). The equivalent mutations R578R579R581A (all three of the indicated Arg residues mutated to Ala) and R585R587R590A were introduced into rabbit muscle glycogen synthase. Whether expressed transiently in COS-1 cells or produced in and purified from Escherichia coli, both mutant enzymes were insensitive to activation by glucose-6-P. The effect of phosphorylation was studied in two ways. Purified, recombinant glycogen synthase was directly phosphorylated by casein kinase 2 and glycogen synthase kinase 3, under conditions that inactivate the wild-type enzyme. In addition, phosphorylation sites were converted to Ala by mutagenesis in wild-type and in the glucose-6-P desensitized mutants expressed in COS-1 cells. Phosphorylation inactivated the R578R579R581A mutant but had little effect on the R585R587R590A. This result was surprising since phosphorylation had the opposite effects on the corresponding yeast enzyme mutants. The results confirm that the region of glycogen synthase, Arg-578-Arg-590, is required for activation by glucose-6-P and suggest that it is part of a sensitive and critical switch involved in transitions between different conformational states. However, the role must differ subtly between the mammalian and the yeast enzymes. (C) 2001 Elsevier Science.