NEW INSIGHTS INTO ENZYME STRUCTURE AND FUNCTION

为了了解疾病的分子基础，特别是 对于涉及单个氨基酸取代的那些，我们需要阐明 蛋白质结构与功能的关系 水平 因此，该项目的长期目标是获得 更深入地了解蛋白质结构与 功能通过使用碱性磷酸酶作为模型系统。 功能 这种酶的作用是催化磷酸盐的非特异性水解 酯，缺乏这种酶的活性会导致致命的 遗传性疾病低磷酸酶，这是由于不足 磷酸盐用于骨骼钙化。 哺乳动物的碱性磷酸酶是 与相应的细菌酶密切相关，并且该酶 已成为研究所有碱性 磷酸酶 本提案的具体目的是回答 结构之间关系的基本问题 和碱性磷酸酶的功能。 我们将集中精力 分子细节的催化机制，需要保持一个 二聚体结构的酶的正确功能，模式， 在亚基之间传递的信息， 基因间互补的关键因素，正确的第二次 结构的形成，金属在催化中的作用， 活性位点周围静电场的贡献， 催化作用 为了实现这一目标，我们将利用 一组数千个点突变产生的碱性 磷酸酶基因的早期努力证明， 该基因和由其产生的蛋白质共线。 这套 今天的突变体为研究提供了独特的资源 碱金属结构与功能的相互关系 磷酸酶。 分析这组突变体以及产生的突变体 在最后一个赠款期间，将通过各种 技术，包括X射线晶体学，31 P和113 Cd NMR，18（O） 同位素效应，以及膦酸盐、硫代磷酸酯的动力学研究 和手性（Rp）[O 16，O 17，O 18]对硝基苯磷酸酯。 相关性 将在氨基酸诱导的功能变化之间进行 取代和突变酶的三维结构。 这项工作不仅对理解这一点很重要， 这是一个特定的系统，但更重要的是制定一般概念 关于酶催化，以及金属在蛋白质中的功能， 为基因间互补提供了分子解释。

In order to understand the molecular basis of diseases, particularly those involving single amino acid substitutions, we need to elucidate the relationship between protein structure and function at the molecular level. Therefore, the long-term goals of this project are to acquire a deeper understanding of the relationship between protein structure and function by using alkaline phosphatase as a model system. The function of this enzyme is to catalyze the nonspecific hydrolysis of phosphate esters, and the lack of the activity of this enzyme results in the fatal hereditary disease hypophosphatasia, which is due to insufficient phosphate for bone calcification. Alkaline phosphatase from mammals is closely related to the corresponding bacterial enzyme, and the enzyme from Escherichia coli has become the model for the study of all alkaline phosphatases. The specific aims of this proposal are to answer fundamental questions concerning the relationship between the structure and function of alkaline phosphatase. We will concentrate on the molecular details of the catalytic mechanism, the need to maintain a dimeric structure for the correct function of the enzyme, the mode by which information is passed between the subunits, a molecular explanation of intergenic complementation, the factors critical for correct secondary structure formation, the function of the metals in catalysis, and the contribution of the electrostatic field around the active site for catalysis. In order to accomplish this goal, we will take advantage of a set of thousands of point mutations created within the alkaline phosphatase gene more than 30 years ago in an early effort to prove that the gene and the protein produced from it were colinear. This set of mutants today provides a unique resource for the investigation of the interrelationship between the structure and function of alkaline phosphatase. Analysis of this set of mutants as well as mutants created during the last grant period will be accomplished by a variety of techniques including x-ray crystallography, 31P and 113Cd NMR, 18(O) isotope effects, and kinetic studies with phosphonates, phosphorothioates and the chiral (Rp) [O16,O17, O18] p-nitrophenyl phosphate. 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, and the function of metals in proteins, and providing a molecular explanation for intergenic complementation.