PHOSPHORYLATION MODULATES CATALYTIC FUNCTION AND REGULATION IN THE CAMP-DEPENDENT PROTEIN-KINASE
PHOSPHORYLATION MODULATES CATALYTIC FUNCTION AND REGULATION IN THE CAMP-DEPENDENT PROTEIN-KINASE
复制标题
DOI：
10.1021/bi00008a007
复制
发表时间：
1995-02-28
期刊：
影响因子：
2.9
通讯作者：
TAYLOR, SS
中科院分区：
文献类型：
--
作者：
ADAMS, JA;MCGLONE, ML;TAYLOR, SS
定点突变被用来删除一个关键的磷酸化位点，Thr-197，附近的cAMP依赖性蛋白激酶的催化亚基的活性位点。该残基存在于许多蛋白激酶中，其磷酸化在很大程度上影响催化活性。我们将Thr-197改变为天冬氨酸和丙氨酸，并测量这些取代对动力学机制和抑制剂亲和力的影响。突变体表达为游离催化亚基和谷胱甘肽-S-转移酶的可溶性融合蛋白。所有三种突变体的K-ATP和K-肽的值相对于野生型酶提高了约2个数量级。粘度测量表明，K-肽的升高是磷酰基转移速率降低而不是底物亲和力降低的结果。这意味着含有磷酸苏氨酸的环，即活化环，不像对cdk 2激酶的无活性形式所提出的那样减少对底物位点的接近[DeBont，H. L.，等人（1993）Nature 363，595-602]和MAP激酶[Zhang，F.，等人（1994）Nature 367，704-711]。突变体与野生型调节亚基缓慢相关，尽管无cAMP的野生型调节亚基化学计量地抑制突变体。一个突变的调节亚基，结合cAMP差，并迅速抑制野生型催化亚基不抑制突变蛋白。这些数据表明，磷酸苏氨酸区域作为一个对接表面的调节亚基在全酶复合物。在生理条件下，突变体的催化效率降低多达500倍，这意味着激活环中的这种和其他蛋白激酶的磷酸化提供了酶功能的分子开关。
Site-directed mutagenesis was used to remove a critical phosphorylation site, Thr-197, near the active site of the catalytic subunit of cAMP-dependent protein kinase. This residue is present in a number of protein kinases, and its phosphorylation largely influences catalytic activity. We changed Thr-197 to aspartic acid and alanine and measured the effects of these substitutions on the kinetic mechanism and inhibitor affinities. The mutants were expressed as the free catalytic subunit and as soluble fusion proteins of glutathione-S-transferase. The values for K-ATP and K-peptide for all three mutants are raised by approximately 2 orders of magnitude relative to the wild-type enzyme. Viscosometric measurements indicate that elevations in K-peptide are the result of reduced rates for phosphoryl transfer and not reduced substrate affinities. This implies that the loop that contains the phosphothreonine, the activation loop, does not reduce access to the substrate site as proposed for the inactive forms of cdk2 kinase [DeBont, H. L., et al. (1993) Nature 363, 595-602] and MAP kinase [Zhang, F., et al. (1994) Nature 367, 704-711]. The mutants associate slowly with the wild-type regulatory subunit, although the cAMP-free wildtype regulatory subunit inhibits the mutants stoichiometrically. A mutant regulatory subunit that binds cAMP poorly and rapidly inhibits the wild-type catalytic subunit does not inhibit the mutant proteins. These data suggest that the phosphothreonine region serves as a docking surface for the regulatory subunit in the holoenzyme complex. Under physiological conditions, the catalytic efficiency of the mutants is lowered by as much as 500-fold, implying that phosphorylation of this and other protein kinases in the activation loop provides a molecular switch for enzymic function.