Glass D B, Krebs E G
J Biol Chem. 1982 Feb 10;257(3):1196-200.
Analogs of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B were used to assess the substrate specificity of cGMP-dependent protein kinase. cGMP-dependent kinase phosphorylated the oligopeptide Arg-Lys-Arg-Ser32-Arg-Lys-Glu with favorable kinetic parameters as compared to those for cAMP-dependent kinase (Glass, D. B., and Krebs, E. G. (1979) J. Biol. Chem. 254, 9728-9738). The contribution of each amino acid to the ability of the peptide to be phosphorylated by cGMP-dependent or cAMP-dependent kinase was studied by replacement of individual residues and evaluation of the kinetic constants of the substituted peptides. Peptides containing acetylated lysine residues or nitroarginine residues were poor substrates for both kinases. Substitution of either arginine 29 or lysine 30 with alanine increased the Km values and decreased the Vmax values for both kinases. Substitution of lysine 34 with alanine increased the Vmax values for both kinases but did not affect the Km values for either enzyme. Substitution of the phosphorylatable serine with a threonine residue greatly depressed the Vmax for both kinases. Peptides in which arginine 31 or arginine 33 were replaced by an alanine residue revealed several apparent differences in the specificity requirements between cGMP-dependent and cAMP-dependent kinases.
与组蛋白H2B磷酸化位点周围序列对应的合成七肽底物类似物被用于评估环磷酸鸟苷（cGMP）依赖性蛋白激酶的底物特异性。与环磷酸腺苷（cAMP）依赖性激酶相比，cGMP依赖性激酶以良好的动力学参数使寡肽精氨酸-赖氨酸-精氨酸-丝氨酸32-精氨酸-赖氨酸-谷氨酸磷酸化（格拉斯，D.B.，和克雷布斯，E.G.（1979年）《生物化学杂志》254，9728 - 9738）。通过替换单个残基并评估取代肽的动力学常数，研究了每个氨基酸对该肽被cGMP依赖性或cAMP依赖性激酶磷酸化能力的贡献。含有乙酰化赖氨酸残基或硝基精氨酸残基的肽对两种激酶来说都是不良底物。用丙氨酸取代精氨酸29或赖氨酸30会增加两种激酶的米氏常数（Km）值并降低最大反应速度（Vmax）值。用丙氨酸取代赖氨酸34会增加两种激酶的Vmax值，但不影响任何一种酶的Km值。用苏氨酸残基取代可磷酸化的丝氨酸会极大地降低两种激酶的Vmax。精氨酸31或精氨酸33被丙氨酸残基取代的肽在cGMP依赖性和cAMP依赖性激酶的特异性要求方面显示出几个明显差异。
