NEW INSIGHTS INTO ENZYME STRUCTURE AND FUNCTION

包括镰状细胞性贫血、β- 地中海贫血、黑蒙性贫血和苯丙酮尿症是由于 一个特定的氨基酸结构中的单个氨基酸改变 蛋白质或酶。 虽然我们可以确定一个 蛋白质到原子分辨率，我们仍然不明白， 细节，这个结构与特定的功能有关。 酶的蛋白质 为了了解 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，我们认为， 该项目的长期目标是获得更深层次的 了解蛋白质结构与 使用E. coli碱性磷酸酶作为模型系统。 这种酶催化磷酸盐的非特异性水解 酯，是所有碱性研究的模型 磷酸酶 该提案的具体目标是回答基本问题， 关于结构与 碱性磷酸酶的功能。 我们将集中精力 催化机制的分子细节， 信息在酶的亚基之间传递， 金属在这种酶中的作用。 我们将使用各种 分子生物学技术来制造 具有单个氨基酸取代的酶。 首先，工作将 专注于分析已经被 创造 氨基酸的额外位点的选择 替代将基于所有的生物化学和结构 数据目前可用。 动力学和生物物理方法，例如 停流动力学，圆二色谱，NMR光谱，和 X射线晶体学将被用来分析的结果， 氨基酸取代。 相关性将在 由氨基酸取代引起的功能变化和 突变酶的三维结构。 这项工作将 不仅对理解这个特殊的 系统，但更重要的是制定一般概念， 酶的催化作用、协同作用以及金属在 proteins.

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.