MUTAGENESIS STUDIES OF THE PHOSPHORYLATION SITES OF RECOMBINANT HUMAN PYRUVATE-DEHYDROGENASE - SITE-SPECIFIC REGULATION
MUTAGENESIS STUDIES OF THE PHOSPHORYLATION SITES OF RECOMBINANT HUMAN PYRUVATE-DEHYDROGENASE - SITE-SPECIFIC REGULATION
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DOI：
10.1074/jbc.270.24.14297
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发表时间：
1995-06-16
影响因子：
4.8
通讯作者：
PATEL, MS
中科院分区：
文献类型：
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作者：
KOROTCHKINA, LG;PATEL, MS
哺乳动物丙酮酸脱氢酶（α（2）β（2））（E（1））由E（1）-激酶和磷酸-E（1）-磷酸酶催化的磷酸化-去磷酸化调节。通过对E（1）α上的三个磷酸化位点（位点1、2和3）进行定点突变，通过将Ser变为Ala，制备了几种具有单突变、双突变和三突变的人E（1）突变体。位点1的突变而不是位点2和/或3的突变降低了E（1）的比活性，并且还增加了硫胺素焦磷酸盐和丙酮酸盐的Km值。E（1）突变体中的位点1、2和3被二氢硫辛酰胺乙酰转移酶-蛋白X-E（1）激酶单独磷酸化或在其他位点存在下磷酸化，表明磷酸化的位点非依赖性机制。每个位点的磷酸化导致E（1）的完全失活。然而，磷酸化和失活的速率是位点特异性的。位点1、2和3被磷酸-E（1）-磷酸酶单独或在其他位点存在下去磷酸化，导致E（1）完全再活化。位点1、2和3的去磷酸化和再活化速率相似，表明随机去磷酸化机制。
Mammalian pyruvate dehydrogenase (alpha(2) beta(2)) (E(1)) is regulated by phosphorylation-dephosphorylation, catalyzed by the E(1)-kinase and the phospho-E(1)-phosphatase. Using site-directed mutagenesis of the three phosphorylation sites (sites 1, 2, and 3) on E(1) alpha, several human E(1) mutants were made with single, double, and triple mutations by changing Ser to Ala, Mutation at site 1 but not at sites 2 and/or 3 decreased E(1) specific activity and also increased K-m values for thiamin pyrophosphate and pyruvate. Sites 1, 2, and 3 in the E(1) mutants were phosphorylated either individually or in the presence of the other sites by the dihydrolipoamide acetyltransferase-protein X-E(1) kinase indicating a site-independent mechanism of phosphorylation. Phosphorylation of each site resulted in complete inactivation of the E(1). However, the rates of phosphorylation and inactivation were site-specific. Sites 1, 2, and 3 were dephosphorylated either individually or in the presence of the other sites by the phospho-E(1)-phosphatase resulting in complete reactivation of the E(1). The rates of dephosphorylation and reactivation were similar for sites 1, 2, and 3, indicating a random dephosphorylation mechanism.