NEW INSIGHTS INTO ENZYME STRUCTURE/FUNCTION

为了阐明疾病的分子基础， 是需要获得一个基本的理解之间的关系， 蛋白质的结构和功能。 这些知识将 让我们更好地理解酶的催化作用， 设计特异性抑制剂来调节酶的活性。 模型 用于该项目的系统是碱性磷酸酶，一种 催化磷酸酯的非特异性水解。 缺乏活动 这种酶的过量会导致致命的遗传性疾病低磷酸酶症， 这是由于磷酸盐不足导致骨钙化。 此外，本发明还提供了一种方法， 碱性磷酸酶和丝氨酸/苏氨酸磷酸酶，它们参与了 代谢控制大量重要的细胞过程，具有 在其机制中的共同中间体。 我们选择了碱性的 磷酸酶;因为这个系统不仅提供了 它本身很容易进行时间分辨的蛋白质晶体学研究，而且 提供了一个独特的系统，其中t调查基本问题 关于蛋白质结构和功能之间的关系。 本修订提案的具体目标是：（i）结合使用 结晶技术结合明智的选择pH值和 温度，以确定酶的三维结构， 在1.75埃处不存在和存在底物，以及 共价和非共价酶磷酸盐复合物，从而揭示了微妙的 详细介绍了反应机理中的每一步，（ii）确定 酶的结构与一系列抑制剂结合，以发展 抑制整个金属磷酸酶类的一般规则， (iii)阐明单个活性位点迁移率的重要性 残基，（iv）继续研究基因内的分子基础 （五）确定突变体的结构，其中活性互补， 为了了解更多关于磷酸酯的信息， 在没有磷酸丝氨酸中间体的情况下水解，和（vi）继续， 阐明单个氨基酸和金属对 结构稳定和催化作用。 此外，晶体学 数据将被用来（我）创建一个逐弗拉姆的电影显示如何这 原型磷酸酶在分子水平上的功能，以及（ii）发展 领导第二代金属磷酸酶抑制剂的设计。

DESCRIPTION: In order to elucidate the molecular basis of diseases, there is a need to acquire a fundamental understanding of the relationship between protei structure and function at the molecular level. This knowledge will allow us to understand enzyme catalysis better, and make it possible to design specific inhibitors that can regulate enzyme activity. The model system to be used for this project is alkaline phosphatase, an enzyme that catalyzes the nonspecific hydrolysis of phosphate esters. Lack of activity of this enzyme results in the fatal hereditary disease hypophosphatasia, which is due to insufficient phosphate for bone calcification. In addition, alkaline phosphatase and the Ser/Thr phosphatases, which are involved in the metabolic control of a large number of important cellular processes, have a common intermediate in their mechanisms. We have selected the alkaline phosphatase from E. coli for this project because this system not only lends itself readily to time-resolved protein crystallographic studies, but also provides a unique system in which t investigate fundamental questions concerning the relationship between protein structure and function. The specific aims of this revised proposal are to (i) use a combination of crystallographic techniques in combination with judicious choice of pH and temperature to determine the three-dimensional structures of the enzyme in the absence and presence of substrates at 1.75 angstroms, as well as the covalent and noncovalent enzyme-phosphate complexes, thus revealing subtle details abou each step in the reaction mechanism, (ii) determine the structures of the enzyme with a series of inhibitors bound so as to develop general rules for inhibition of the entire class of metallophosphatases, (iii) elucidate the importance of mobility of individual active site residues, (iv) continue to investigate the molecular basis of intragenic complementation, (v) determine the structure of mutants in which the active site serine has been replaced in order to learn more about phosphoester hydrolysis without a phosphoserine intermediate, and (vi) continue to elucidate the contribution of individual amino acids and the metals towards structural stabilization and catalysis. In addition, the crystallographic data will be used to (I) create a frame-by-fram movie showing how this prototypical phosphatase functions at the molecular level and (ii) develop leads for the design of second generation metallophosphatase inhibitors.