NEW INSIGHTS INTO ENZYME STRUCTURE AND FUNCTION

各种疾病，包括镰状细胞性贫血，贝塔- 地中海贫血、泰-萨克氏症和苯丙酮尿症是由 特定结构中的单一氨基酸变化 蛋白质或酶。虽然我们可以确定一个 蛋白质到原子的分辨率，我们仍然不知道是如何，在 细节，这种结构是与特定的功能相关的 酶的蛋白质。为了更好地理解分子基础 疾病，我们需要在分子水平上阐明 蛋白质结构与功能的关系。因此， 这个项目的长期目标是获得更深层次的 对蛋白质结构与蛋白质之间关系的认识 以大肠杆菌碱性磷酸酶为模型系统进行功能研究。 这种酶催化磷酸盐的非特异性水解。 酯类，是研究所有碱性物质的模型 磷酸酶。 这项提议的具体目的是回答根本问题 关于结构与结构关系的几个问题 碱性磷酸酶的功能。我们将专注于 催化机理、催化方式的分子细节 信息在酶的亚基之间传递，而 金属在这种酶中的作用。我们将使用各种 分子生物学技术来创造改变版本的 具有单一氨基酸取代的酶。最初，工作将 专注于分析已经存在的突变 已创建。氨基酸附加位点的选择 替换将基于所有的生化和结构 当前可用的数据。动力学和生物物理方法，如 停流动力学、圆二色谱、核磁共振光谱和 将使用X射线结晶学来分析测试结果 氨基酸替换。将在两个数据之间建立关联 氨基酸替代引起的功能改变和 突变酶的三维结构。这项工作将 不仅对理解这一特殊情况很重要 系统，但更重要的是用来制定关于 酶的催化作用、协同作用及金属在其中的作用 蛋白质。

A variety of diseases including sickle cell anemia, beta- thalassemia, Tay-Sachs and phenylketonuria are the result of a single amino acid alteration in the structure of a particular protein or enzyme. Although we can determine the structure of a protein to atomic resolution, we still do not understand how, in detail, this structure is related to the function of the particular protein of enzyme. In order to understand the molecular basis of diseases, we need to elucidate at the molecular level the relationship between protein structure and function. Therefore, the long term goals of this project are to acquire a deeper understanding of the relationship between protein structure and function by using E. coli alkaline phosphatase as a model system. This enzyme catalyzes the nonspecific hydrolysis of phosphate esters, and is the model for the study of all alkaline phosphatases. The specific aims of this proposal are to answer fundamental questions concerning the relationship between structure and function of alkaline phosphatase. We will concentrate on the molecular details of the catalytic mechanism, the mode by which information is passed between the subunits of the enzyme, and the function of the metals in this enzyme. We will use a variety of molecular biology techniques to create altered versions of the enzyme with single amino acid substitutions. Initially, work will concentrate on the analysis of mutants that have been already created. Selection of additional sites for amino acid substitutions will be based on all the biochemical and structural data currently available. Kinetic and biophysical methods such as stopped-flow kinetics, circular dichroism, NMR spectroscopy, and X-ray crystallography will be used to analyze the results of the amino acid substitutions. Correlations will be made between the functional changes induced by the amino acid substitution and the three-dimensional structure of the mutant enzymes. This work will not only be important for the understanding of this particular system, but more importantly for formulating general concepts about enzyme catalysis, cooperativity and the function of metals in proteins.