Glass D B, Krebs E G
J Biol Chem. 1979 Oct 10;254(19):9728-38.
The substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinases have been compared by kinetic analysis using synthetic peptides as substrates. Both enzymes catalyzed the transfer of phosphate from ATP to calf thymus histone H2B, as well as to two synthetic peptides, Arg-Lys-Arg-Ser32-Arg-Lys-Glu and Arg-Lys-Glu-Ser36-Tyr-Ser-Val, corresponding to the amino acid sequences around serine 32 and serine 36 in histone H2B. Serine 38 in the latter peptide was not phosphorylated by either enzyme. Cyclic GMP-dependent kinase and cyclic AMP-dependent kinase catalyzed the incorporation of 1.1 and 2.0 mol of phosphate/mol of histone H2B, respectively. The phosphorylation of histone H2B, respectively. The phosphorylation of histone H2B by cyclic GMP-dependent kinase showed two distinct optima as the magnesium concentration was increased. However, the phosphorylation of either synthetic peptide by this enzyme was depressed at high magnesium concentrations. As the pH of reaction mixtures was elevated from pH 6 to pH 9, the rate of phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase continually increased. Acetylation of the NH2 terminus of the peptide did not qualitatively affect this pH profile, but did increase the Vmax value of the enzyme 3-fold. The apparent Km and Vmax values for the phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase were 21 microM and 4.4 mumol/min/mg, respectively. The synthetic peptide Arg-Lys-Glu-Ser36-Tyr-Ser-Val was a relatively poor substrate for cyclic GMP-dependent kinase, exhibiting a Km value of 732 microM, although the Vmax was 12 micromol/min/mg. With histone H2B as substrate for the cyclic GMP-dependent kinase, two different Km values were apparent. The Km values for cyclic AMP-dependent kinase for either synthetic peptide were approximately 100 microM, but the Vmax for Arg-Lys-Arg-Ser32-Arg-Lys-Glu was 1.1 mumol/min/mg, while the Vmax for Arg-Lys-Glu-Ser36-Tyr-Ser-Val was 16.5 mumol/min/mg. These data suggest that although the two cyclic nucleotide-dependent protein kinases have similar substrate specificities, the determinants dictated by the primary sequence around the two phosphorylation sites in histone H2B are different for the two enzymes.
通过使用合成肽作为底物的动力学分析，比较了环鸟苷酸依赖性蛋白激酶和环腺苷酸依赖性蛋白激酶的底物特异性。两种酶都催化了磷酸从ATP转移至小牛胸腺组蛋白H2B，以及转移至两种合成肽，即对应于组蛋白H2B中丝氨酸32和丝氨酸36周围氨基酸序列的精氨酸-赖氨酸-精氨酸-丝氨酸32-精氨酸-赖氨酸-谷氨酸和精氨酸-赖氨酸-谷氨酸-丝氨酸36-酪氨酸-丝氨酸-缬氨酸。后一种肽中的丝氨酸38未被任何一种酶磷酸化。环鸟苷酸依赖性激酶和环腺苷酸依赖性激酶分别催化每摩尔组蛋白H2B掺入1.1摩尔和2.0摩尔磷酸。环鸟苷酸依赖性激酶对组蛋白H2B的磷酸化随着镁离子浓度增加呈现出两个不同的最佳值。然而，该酶对任何一种合成肽的磷酸化在高镁离子浓度下受到抑制。随着反应混合物的pH从pH 6升高至pH 9，环鸟苷酸依赖性激酶对精氨酸-赖氨酸-精氨酸-丝氨酸32-精氨酸-赖氨酸-谷氨酸的磷酸化速率持续增加。肽的氨基末端乙酰化在性质上不影响该pH曲线，但确实使酶的Vmax值增加了3倍。环鸟苷酸依赖性激酶对精氨酸-赖氨酸-精氨酸-丝氨酸32-精氨酸-赖氨酸-谷氨酸磷酸化的表观Km和Vmax值分别为21 microM和4.4 mumol/min/mg。合成肽精氨酸-赖氨酸-谷氨酸-丝氨酸36-酪氨酸-丝氨酸-缬氨酸是环鸟苷酸依赖性激酶相对较差的底物，其Km值为732 microM，尽管Vmax为12 micromol/min/mg。以组蛋白H2B作为环鸟苷酸依赖性激酶的底物时，出现了两个不同的Km值。环腺苷酸依赖性激酶对任何一种合成肽的Km值约为100 microM，但对精氨酸-赖氨酸-精氨酸-丝氨酸32-精氨酸-赖氨酸-谷氨酸的Vmax为1.1 mumol/min/mg，而对精氨酸-赖氨酸-谷氨酸-丝氨酸36-酪氨酸-丝氨酸-缬氨酸的Vmax为16.5 mumol/min/mg。这些数据表明，尽管两种环核苷酸依赖性蛋白激酶具有相似的底物特异性，但组蛋白H2B中两个磷酸化位点周围一级序列所决定的决定因素对于这两种酶是不同的。
