3D STRUCTURE OF THE CYCLIC AMP DEPENDENT PROTEIN KINASE

蛋白激酶是关键的调节分子，参与了许多 生化途径。作为催化剂，这些酶可以放大细胞内或 细胞外信号。由于它们宽广而又有限的底物 特异性，它们能够一次调节不止一种底物蛋白 时间，协调细胞内新陈代谢。了解蛋白质是如何 激酶实现调节细胞过程所必需的特异性是一种 这是生物学中的一个重要问题，也是这项提议的长期目标。 具体地说，对cAMP依赖的TPK1进行了详细的结构研究 提出了酿酒酵母的蛋白激酶催化亚基。 现有的衍射到2.2安分辨率的TPK1晶体将是 用重原子化合物衍生化，解决晶相 问题，并产生了它的三维结构。该结构将 揭示先前证明涉及的关键残基的位置 在底物识别和催化方面，并揭示了一个 蛋白激酶结构域。这项工作将通过预测 底物上与TPK1不同的其他蛋白激酶的结构 特异性采用比较建模的方法。由此产生的预测 蛋白激酶底物的选择性将在体外进行测试 诱变。嵌合激酶将被合成并表征为 将计算机建模预测的结构特征与酶联系起来 活动。对蛋白激酶结构的全面了解将加快 可能在治疗中有用的特定抑制剂的开发 肿瘤性疾病。

Protein kinases are key regulatory molecules that participate in many biochemical pathways. As catalysts, these enzymes can amplify intra- or extracellular signals. By virtue of their broad yet limited substrate specificity, they are able to regulate more than one substrate protein at a time, coordinating intracellular metabolism. Understanding how protein kinases achieve the specificity necessary to regulate cell processes is an important problem in biology, and is the long-term goal of this proposal. Specifically, a detailed structural study of TPK1, the cAMP-dependent protein kinase catalytic subunit of Saccharomyces cerevisiae, is proposed. Existing crystals of TPK1 that diffract to 2.2 A resolution will be derivatized with heavy atom compounds, solving the crystallographic phase problem, and yielding its three-dimensional structure. The structure will reveal the location of key residues previously demonstrated to be involved in substrate recognition and catalysis, and reveal the overall folding of a protein kinase domain. This work will be extended by predicting the structure of other protein kinases that differ from TPK1 in substrate specificity using comparative modeling methods. The resulting prediction of protein kinase substrate selectively will be tested by in vitro mutagenesis. Chimeric kinases will be synthesized and characterized to relate the structural features predicted by computer modeling to enzymatic activity. A complete understanding of protein kinase structure will speed the development of specific inhibitors potentially useful in the treatment of neoplastic disease.