Weijland A, Neubauer G, Courtneidge S A, Mann M, Wierenga R K, Superti-Furga G
European Molecular Biology Laboratory, Heidelberg, Germany.
Eur J Biochem. 1996 Sep 15;240(3):756-64. doi: 10.1111/j.1432-1033.1996.0756h.x.
The catalytic domain of chicken Src including the C-terminal tail (Src-CD), has been expressed in Schizosaccharomyces pombe and purified to homogeneity. The expressed protein is a mixture of unphosphorylated (80%) and mono-phosphorylated (20%) species, that can be separated from each other by Mono Q chromatography. By a novel mass spectrometric method that utilizes parent ion scans of unseparated peptide mixtures, we found that the mono-phosphorylated form is phosphorylated either at Tyr416 or at Tyr436. The stability of Src-CD is comparable to the wild-type protein. Src-CD auto-phosphorylates and efficiently phosphorylates substrate peptides and proteins. Auto-phosphorylation occurs by an intermolecular mechanism and is completely inhibited by an excess of substrate peptide. Kinetic measurements for two exogenous substrates, the Src substrate peptide (AEEEIYGEFEAKKKK) and denatured enolase, showed that the overall activity (kcat) of the Src-CD molecule is about 10 times higher than that of wild-type Src. The kcat values for phosphorylation of the Src substrate peptide are similar for the unphosphorylated and monophosphorylated Src-CD (50 min-1), but the apparent K(m) values differ significantly (approximately 3 microM and 10 microM, respectively). Therefore, at low substrate concentrations in vitro the mono-phosphorylated form is more active, in agreement with the importance of Tyr416 for in vivo activity. The apparent K(m) values of the mono-phosphorylated Src-CD and wild-type Src for the Src substrate peptide and enolase are similar, indicating that, under these conditions, the kinase domain is mainly responsible for substrate binding.
包含C末端尾巴的鸡源Src催化结构域（Src-CD）已在粟酒裂殖酵母中表达并纯化至同质。表达的蛋白质是未磷酸化（80%）和单磷酸化（20%）物种的混合物，可通过Mono Q色谱法彼此分离。通过一种利用未分离肽混合物的母离子扫描的新型质谱方法，我们发现单磷酸化形式在Tyr416或Tyr436处被磷酸化。Src-CD的稳定性与野生型蛋白相当。Src-CD能自身磷酸化并有效磷酸化底物肽和蛋白质。自身磷酸化通过分子间机制发生，并被过量的底物肽完全抑制。对两种外源底物，即Src底物肽（AEEEIYGEFEAKKKK）和变性烯醇化酶的动力学测量表明，Src-CD分子的总体活性（kcat）比野生型Src高约10倍。未磷酸化和单磷酸化的Src-CD对Src底物肽磷酸化的kcat值相似（50 min-1），但表观K(m)值有显著差异（分别约为3 microM和10 microM）。因此，在体外低底物浓度下，单磷酸化形式更具活性，这与Tyr416对体内活性的重要性一致。单磷酸化的Src-CD和野生型Src对Src底物肽和烯醇化酶的表观K(m)值相似，表明在这些条件下，激酶结构域主要负责底物结合。
