Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.
Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.
复制标题
哺乳动物丙酮酸脱氢酶激酶和丙酮酸脱氢酶磷酸酶的合成肽底物。
DOI：
10.1016/0003-9861(85)90543-0
复制
发表时间：
1985
影响因子：
3.9
通讯作者：
Reed,LJ
中科院分区：
文献类型：
--
作者：
Mullinax,TR;Stepp,LR;Brown,JR;Reed,LJ
丙酮酸脱氢酶激酶和丙酮酸脱氢酶的特异性磷酸酶是探索使用合成肽序列对应的网站磷酸化对丙酮酸脱氢酶1和2(酪氨酸他g他Ser (P1)遇到SerAspProgValSer (P2)酪氨酸Arg)。在第8位含有天冬氨酸的去磷酸四肽比在第8位含有天冬酰胺的四肽是更好的激酶底物。这两种肽的表观kmandvv分别为0.43和6.1,2.7和2.4 nmol的32p掺入/ min/mg。天冬氨酸残基的甲基化也使四肽的表观分子量增加了约14倍。这些结果表明，在磷酸化位点1的羧基末端的酸性残基是激酶特异性的重要决定因素。磷酸仅被激酶结合到合成肽的1号位点。磷酸酶对含天冬氨酸的磷酸化四肽的表观kmo为0.28，avo为2.3 μmol 32p /min/mg。天冬氨酸残基甲基化对去磷酸化无显著影响。甲酸裂解天冬氨酸-丙氨酸键产生的八肽和磷酸肽分别比四肽和磷酸四肽对激酶和磷酸酶的底物更差。通过乙酰化或赖氨酸修饰氨基末端对激酶和磷酸酶活性只有轻微的影响。
The specificities of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase were probed using synthetic peptides corresponding to the sequence around phosphorylation sites 1 and 2 on pyruvate dehydrogenase [TyrHisGlyHisSer(P1)Met SerAspProGlyValSer(P2)TyrArg]. The dephosphotetradecapeptide containing aspartic acid at position 8 was a better substrate for the kinase than was the tetradecapeptide containing asparagine at position 8. The apparentKmandVvalues for the two peptides were 0.43 and 6.1 mmand 2.7 and 2.4 nmol of32P incorporated/ min/mg, respectively. Methylation of the aspartic acid residue also increased the apparentKmof the tetradecapeptide about 14-fold. These results indicate that an acidic residue on the carboxyl-terminal side of phosphorylation site 1 is an important specificity determinant for the kinase. Phosphate was incorporated only into site 1 of the synthetic peptide by the kinase. The phosphatase exhibited an apparentKmof 0.28 mmand aVof 2.3 μmol of32P released/min/mg for the phosphorylated tetradecapeptide containing aspartic acid. Methylation of the aspartic acid residue had no significant effect on dephosphorylation. The octapeptide and phosphooctapeptide produced by cleavage of the aspartyl-prolyl bond by formic acid were poorer substrates for the kinase and phosphatase than were the tetradecapeptide and phosphotetradecapeptide, respectively. Modification of the amino terminal by acetylation or lysine addition had only a slight effect on the kinase and phosphatase activities.