STRUCTURAL BASIS FOR THE LOW AFFINITIES OF YEAST CAMP-DEPENDENT AND MAMMALIAN CGMP-DEPENDENT PROTEIN-KINASES FOR PROTEIN-KINASE INHIBITOR PEPTIDES
STRUCTURAL BASIS FOR THE LOW AFFINITIES OF YEAST CAMP-DEPENDENT AND MAMMALIAN CGMP-DEPENDENT PROTEIN-KINASES FOR PROTEIN-KINASE INHIBITOR PEPTIDES
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DOI：
10.1021/bi00121a021
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发表时间：
1992-02-18
期刊：
影响因子：
2.9
通讯作者：
WALSH, DA
中科院分区：
文献类型：
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作者：
GLASS, DB;FELLER, MJ;WALSH, DA
测定了酿酒酵母cAMP依赖性蛋白激酶和哺乳动物cGMP依赖性蛋白激酶的催化亚基（C1）对蛋白激酶抑制剂（PKI）肽PKI（6-22）酰胺和7种类似物的亲和力。这些类似物在N-末端α-螺旋、C-末端假底物部分或PKI肽的中心连接区域中含有结构改变。在所有情况下，PKI肽作为酵母C1的抑制剂比作为哺乳动物C-alpha亚基的抑制剂活性明显降低。酵母酶的K（i）值比哺乳动物酶高5- 290倍。与这些结果一致，酵母C1对肽底物Kemptide表现出较高的K（m）。所有的PKI肽对哺乳动物cGMP依赖性蛋白激酶的活性甚至低于对酵母cAMP依赖性蛋白激酶的活性，并且Kemptide是前一种酶的较差底物。这些同源蛋白激酶的氨基酸序列在已知与PKI肽中的决定簇相互作用的哺乳动物C-α亚基的活性位点中的残基周围的比对[Edghton，D. R.，郑杰，滕艾克湖F.、Xuong，N-h，Taylor，S.美国，& Sowadski，J. M.（1991）Science 253，414-420]提供了酵母Cl和cGMP依赖性蛋白激酶对结合肽抑制剂和底物的固有较低亲和力的结构基础。酵母cAMP依赖性蛋白激酶和哺乳动物cGMP依赖性蛋白激酶都缺少与PKI的假底物部分中的精氨酸-18相互作用的三个酸性残基中的两个。此外，cGMP依赖性蛋白激酶似乎完全缺乏识别PKI肽的N-末端中的重要苯丙氨酸-10残基的疏水/芳香口袋，并且酵母蛋白激酶在该位点处对抑制剂的结合似乎部分受损。
Affinities of the catalytic subunit (C1) of Saccharomyces cerevisiae cAMP-dependent protein kinase and of mammalian cGMP-dependent protein kinase were determined for the protein kinase inhibitor (PKI) peptide PKI(6-22)amide and seven analogues. These analogues contained structural alterations in the N-terminal alpha-helix, the C-terminal pseudosubstrate portion, or the central connecting region of the PKI peptide. In all cases, the PKI peptides were appreciably less active as inhibitors of yeast C1 than of mammalian C-alpha subunit. K(i) values ranged from 5- to 290-fold higher for the yeast enzyme than for its mammalian counterpart. Consistent with these results, yeast C1 exhibited a higher K(m) for the peptide substrate Kemptide. All of the PKI peptides were even less active against the mammalian cGMP-dependent protein kinase than toward yeast cAMP-dependent protein kinase, and Kemptide was a poorer substrate for the former enzyme. Alignment of amino acid sequences of these homologous protein kinases around residues in the active site of mammalian C-alpha subunit known to interact with determinants in the PKI peptide [Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N-h, Taylor, S. S., & Sowadski, J. M. (I 99 1) Science 253, 414-420] provides a structural basis for the inherently lower affinities of yeast C1 and cGMP-dependent protein kinase for binding peptide inhibitors and substrates. Both yeast cAMP-dependent and mammalian cGMP-dependent protein kinases are missing two of the three acidic residues that interact with arginine-18 in the pseudosubstrate portion of PKI. Further, the cGMP-dependent protein kinase appears to completely lack the hydrophobic/aromatic pocket that recognizes the important phenylalanine-10 residue in the N-terminus of the PKI peptide, and binding of the inhibitor by the yeast protein kinase at this site appears to be partially compromised.