NEW INSIGHTS INTO ENZYME STRUCTURE/FUNCTION

描述：为了阐明疾病的分子基础，有 是需要从根本上理解 蛋白质在分子水平上的结构和功能。这一知识将会 让我们更好地了解酶的催化作用，并使之有可能 设计可以调节酶活性的特定抑制剂。模型 这个项目使用的系统是碱性磷酸酶，一种可以 催化磷酸酯的非特异性水解。缺乏活动 这种酶会导致致命的遗传性疾病低磷酸盐血症， 这是由于骨钙化所需的磷酸盐不足。此外, 碱性磷酸酶和丝氨酸/苏氨酸磷酸酶，它们参与了 代谢控制的大量重要的细胞过程，有一个 在它们的机制中有共同的中间体。我们选择了碱性的 从大肠杆菌中提取磷酸酶用于这个项目，因为这个系统不仅可以 容易进行时间分辨蛋白质结晶学研究，但也 提供了一个独特的系统，在该系统中t调查基本问题。 关于蛋白质结构和功能之间的关系。 这项修订提案的具体目标是：(I)结合使用 结晶学技术与明智地选择pH和 温度对酶的三维结构的影响 1.75埃时衬底的存在和不存在，以及 共价和非共价的酶-磷酸复合体，从而揭示了微妙的 详细说明反应机理中的每一步，(Ii)确定 与一系列抑制剂结合的酶的结构 抑制整类金属磷酸酶的一般规则， (Iii)阐明个别活跃地点移动性的重要性 残基，(Iv)继续研究基因内的分子基础 互补作用，(V)决定突变体的结构，在突变体中活性 为了了解更多关于磷酸酯的信息，已替换了站点丝氨酸 没有磷酸丝氨酸中间体的水解，以及(Vi)继续 阐明单个氨基酸和金属对 结构稳中有催化。此外，结晶学研究表明， 数据将被用来(I)创建一帧接一帧的电影，展示这是如何 典型的磷酸酶在分子水平上的功能和(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.