Klingbeil C K, Gill G N, Cadena D L
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla 92093-0650.
Arch Biochem Biophys. 1995 Feb 1;316(2):745-50. doi: 10.1006/abbi.1995.1099.
The epidermal growth factor receptor contains five autophosphorylation sites in its C-terminal region. Synthetic peptides based on the major autophosphorylation site at Tyr 1173 were tested as substrates of the intracellular domain of the epidermal growth factor receptor. A peptide containing acidic residues N-terminal to the substrate Tyr as well as the Tyr-Met-Xaa-Met motif of the insulin receptor substrate 1 had a Km value of 15 microM, the lowest value for a synthetic peptide reported to date. Another important residue contributing to substrate binding is the Tyr itself, or more specifically, the hydroxyl group of the Tyr. Substituting Phe for Tyr results in a peptide that is ineffective as an inhibitor of kinase phosphorylation. However, substitution of a Ser residue does not restore a functional substrate, indicating specificity for the Tyr hydroxyl. Secondary structure algorithms predicted that the peptide substrate based on the native sequence at Tyr 1173 would have a propensity to adopt a helical conformation in solution. Circular dichroism spectroscopy confirmed this prediction. The secondary structure of the peptide substrate is significant in its consistency with the idea that secondary structure is an important determinant in substrate recognition by protein tyrosine kinases.
表皮生长因子受体在其C末端区域含有五个自身磷酸化位点。基于酪氨酸1173处主要自身磷酸化位点的合成肽被测试作为表皮生长因子受体内结构域的底物。在底物酪氨酸N末端含有酸性残基以及胰岛素受体底物1的酪氨酸-甲硫氨酸-Xaa-甲硫氨酸基序的肽的Km值为15微摩尔，是迄今为止报道的合成肽的最低值。另一个有助于底物结合的重要残基是酪氨酸本身，或者更具体地说，是酪氨酸的羟基。用苯丙氨酸取代酪氨酸会产生一种作为激酶磷酸化抑制剂无效的肽。然而，丝氨酸残基的取代并不能恢复功能性底物，这表明对酪氨酸羟基具有特异性。二级结构算法预测，基于酪氨酸1173处天然序列的肽底物在溶液中倾向于采用螺旋构象。圆二色光谱证实了这一预测。肽底物的二级结构与其二级结构是蛋白质酪氨酸激酶识别底物的重要决定因素这一观点的一致性具有重要意义。
