Deciphering Enzyme Specificity: Amidohydrolase Superfamily

本申请中描述的研究的长期目标是针对全面的 了解与蛋白质结构和底物相关的物理和化学参数 在酶催化反应中的识别。这一目标的目标是发展一般 确定酶的反应和底物专一性的方法和新的方案 未知功能的。对400多万个基因的功能注释的批判性评估 到目前为止已经测序的结果表明，大约三分之一的编码蛋白具有 不确定、未知或/不正确的函数赋值。这一观察结果表明，有相当一部分 新陈代谢多样性的特征仍有待适当的描述。为此，我们将利用计算 将高能中间体对接到未知功能的酶的活性部位模型 确定最有可能的底物。这些努力的补充将是合成和筛选 化学库和从操纵子和基因组环境中提取进一步的代谢信息。 这一目标将通过集中于阐明底物和反应曲线来实现 氨基水解酶超家族中的全部酶。酰胺水解酶超家族是一种 具有大量底物多样性的一组酶，嵌入在被锻造的活性部位中 来自(p/a)8桶的构造褶皱。超过6,000个独特的蛋白质序列已被鉴定为成员 氨基水解酶超家族。这个超家族的成员已经被证明催化水解酶 酰胺、内酯和有机磷酸酯，以及脱羧基、水合和异构化 反应。然而，这个广泛的超级大家庭中的相当一部分成员有一个模棱两可的 底物和反应的特异性仍有待解开。这一超家族酶的成员 包括二氢核酸酶、尿素酶和腺苷脱氨酶。这个特殊的超级家族的标志是 酶是一个(p/a)8桶结构域的C末端的活性部位，它包含一个 单核或双核金属中心，主要但不完全用于激活溶剂 亲核攻击亲电官能团的水。底物与反应多样性 包含在这个酶超家族中将提供对分子机制的独特见解 从现有的结构模板进化和开发新的酶活性。 相关性(见说明)； 本应用程序的总体目标是开发新的和通用的 底物未知的酶的功能的阐明方法。这些努力将揭开新的 并确定治疗干预的新靶点。

The long-term objective for the research described in this application is directed at a comprehensive understanding of the physical and chemical parameters that relate protein structure and substrate recognition in enzyme-catalyzed reactions. This objective is aimed toward the development of general methodologies and novel protocols for the determination of reaction and substrate specificities for enzymes of unknown function. A critical assessment of the functional annotations forthe more than four million genes that have been sequenced to date suggests that approximately one-third of the encoded proteins have an uncertain, unknown, or /ncorrecf functional assignment. This observation suggests that a significant fraction of the metabolic diversity remains to be properly characterized. Toward this end we will utilize computational docking of high energy intermediates to models of the active sites for enzymes of unknown function to identify the most probable substrates. These efforts will be complemented by the synthesis and screening of chemical libraries and the abstraction of further metabolic information from operon and genomic context. This goal will be pursued by concentrating on the elucidation of the substrate and reaction profiles forthe entire ensemble of enzymes within the amidohydrolase superfamily. The amidohydrolase superfamily is a group of enzymes which has a substantial substrate diversity embedded within active sites that are forged from a (p/a)8-barrel structural fold. Over 6,000 unique protein sequences have been identified as members of the amidohydrolase superfamily. Members of this superfamily have been shown to catalyze the hydrolysis of amides, lactones and organophosphate esters, in addition to decarboxylation, hydration, and isomerization reactions. However, a substantial fraction of the members of this broad superfamily have an ambiguous substrate and reaction specificity that remains to be unraveled. Members of this superfamily of enzymes include dihydroorotase, urease, and adenosine deaminase. The hallmark for this particular superfamily of enzymes is an active site at the C-terminal end of a (p/a)8-barrel structural domain that contains a mononuclear or binuclear metal center that functions predominantly, but not exclusively, to activate solvent water for nucleophilic attack on electrophilic functional groups. The substrate and reaction diversity contained within this enzyme superfamily will provide unique insights into the molecular mechanisms for the evolution and development of novel enzymatic activities from existing structural templates. RELEVANCE (See instructions); The overall objective of this application is directed towards the development of novel and general methods for the elucidation of function for enzymes with unknown substrates. These efforts will unveil new metabolic transformations and identify new targets for therapeutic intervention.