Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties of phospho- and dephospho- forms and of two mutants in which Ser32 has been changed by site-directed mutagenesis.
Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties of phospho- and dephospho- forms and of two mutants in which Ser32 has been changed by site-directed mutagenesis.
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DOI：
10.1016/s0021-9258(18)42851-7
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发表时间：
1992-03
期刊：
影响因子：
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通讯作者：
I. Kurland;M. El‐Maghrabi;J. Correia;S. Pilkis
中科院分区：
文献类型：
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作者：
I. Kurland;M. El‐Maghrabi;J. Correia;S. Pilkis
在将 cAMP 依赖性磷酸化位点 (Ser32) 定点突变为天冬氨酸或丙氨酸后，检查了 cAMP 依赖性蛋白激酶催化磷酸化调节 6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶活性的机制。突变型和野生型酶在大肠杆菌的丰富培养基中过表达，水平高达 30 毫克/升，然后纯化至均质。 Ser32-Ala 突变体的动力学特性与去磷酸化的野生型双功能酶相同。 Ser32 突变为天冬氨酸模拟了 cAMP 依赖性磷酸化的几种效应：6-磷酸果糖 2-激酶的 Km 增加，果糖 2,6-二磷酸酶的最大速度增加。 Ser32-Asp突变体的果糖2,6-双磷酸酶活性是磷酸化野生型酶的75%，突变体的激酶反应对果糖6-磷酸具有相同的依赖性，而在较宽的pH范围内其最大速度仅为磷酸化野生型酶的60%。此外，催化亚基催化的 Ser32-Ala 突变体在 Ser33 上的体外磷酸化使 6-磷酸果糖-2-激酶对果糖 6-磷酸的 Km 增加了 4 倍。这些结果支持这样的假设：Ser32 是双功能酶活性调节中的重要残基，并且 Ser32 的磷酸化可以在功能上被天冬氨酸取代。结果表明负电荷在磷酸化作用中发挥作用。
The mechanism by which cAMP-dependent protein kinase-catalyzed phosphorylation modulates the activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was examined after site-specific mutation of the cAMP-dependent phosphorylation site (Ser32) to aspartic acid or alanine. The mutant and wild-type enzymes were overexpressed in Escherichia coli in a rich medium to levels as high as 30 mg/liter and were then purified to homogeneity. The kinetic properties of the Ser32-Ala mutant were identical with the dephosphorylated wild-type bifunctional enzyme. Mutation of Ser32 to aspartic acid mimicked several effects of cAMP-dependent phosphorylation: there was an increase in the Km for fructose 6-phosphate for 6-phosphofructo-2-kinase and an increase in the maximal velocity of fructose-2,6-bisphosphatase. Fructose-2,6-bisphosphatase activity of the Ser32-Asp mutant was 75% that of the phosphorylated wild-type enzyme, the mutant's kinase reaction had an identical dependence on fructose 6-phosphate, while its maximum velocity was only 60% that of the phosphorylated wild-type enzyme over a wide pH range. Furthermore, catalytic subunit-catalyzed in vitro phosphorylation of the Ser32-Ala mutant on Ser33 increased the Km for fructose 6-phosphate by 4-fold for the 6-phosphofructo-2-kinase. The results support the hypothesis that Ser32 is an important residue in the regulation of the activities of the bifunctional enzyme and that phosphorylation of Ser32 can be functionally substituted by aspartic acid. The results suggest a role for negative charge in the effect of phosphorylation.