Adenylosuccinate Lyase: A Study of the Novel Intersubunit Active Sites

腺苷琥珀酸裂解酶：新型亚基间活性位点的研究

• 批准号: 0110878
• 负责人: Roberta Colman
• 金额: $ 40.21万
• 依托单位: University of Delaware
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2004-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0110878&HistoricalAwards=false
• 关键词: Adenylosuccinate; Lyase; Study; Novel; Intersubunit

Adenylosuccinate lyase catalyzes a critical step in purine biosynthesis by converting adenylosuccinate to AMP and fumarate. High-resolution structures of two bacterial enzymes in the absence of substrate have been reported. Three histidines, contributed by two different subunits, have been identified in the active site. However, the structure of adenylosuccinate lyase suggests that in an enzyme tetramer, three subunits actually contribute to each of the four active sites. This project's goal is to understand the major chemical and structural contributions to catalysis by normal adenylosuccinate lyase and the molecular basis for decreased activity in patients with ASL deficiency. The specific aims address three questions: 1. Which additional amino acids of adenylosuccinate lyase contribute to catalysis and/or substrate binding? This issue is approached by site-directed mutagenesis of amino acids postulated as at the active site of B. subtilis adenylosuccinate lyase. Stable analogs of adenylosuccinate, inert to enzymatic cleavage, will be synthesized and used to prepare crystalline enzyme-inhibitor complexes; they will also be used to assess substrate binding by enzymes. 2. How many subunits contribute to each active site of adenylosuccinate lyase? Complementation experiments will be conducted in which pairs of different, inactive mutants are mixed and tested for restoration of activity in hybrid tetramers. 3. What is the molecular basis for the structure-function relations in the adenylosuccinate lyase? B. subtilis adenylosuccinate lyase will be engineered with amino acid substitutions equivalent to those in some human variants with adenylosuccinate lyase deficiency. These mutant enzymes will be models to elucidate further the structural/chemical basis of the enzyme function. (This project is supported jointly by the Molecular Biochemistry and Metabolic Biochemistry Programs.)

琥珀酸腺苷裂解酶催化嘌呤生物合成的一个关键步骤，将琥珀酸腺苷转化为AMP和富马酸。在没有底物的情况下，已经报道了两种细菌酶的高分辨率结构。三个组氨酸，贡献了两个不同的亚基，已确定在活性部位。然而，腺苷琥珀酸裂解酶的结构表明，在酶四聚体中，三个亚基实际上对四个活性位点中的每一个都有贡献。该项目的目标是了解正常腺苷琥珀酸裂解酶催化的主要化学和结构贡献，以及ASL缺乏症患者活性下降的分子基础。具体目标涉及三个问题：1。腺苷琥珀酸裂解酶的哪些氨基酸有助于催化和/或底物结合？这个问题是通过在枯草芽孢杆菌腺苷琥珀酸裂解酶的活性位点对氨基酸进行定点诱变来解决的。将合成对酶裂解惰性的稳定的腺苷琥珀酸类似物，并用于制备晶体酶抑制剂配合物；它们还将用于评估酶对底物的结合。2. 腺苷琥珀酸裂解酶的每个活性位点有多少个亚基？将进行互补实验，其中对不同的失活突变体混合并测试杂交四聚体的活性恢复。3. 腺苷琥珀酸解酶结构-功能关系的分子基础是什么？枯草芽孢杆菌腺苷琥珀酸裂解酶将被改造成与一些人类腺苷琥珀酸裂解酶缺乏变异的氨基酸替换。这些突变酶将成为进一步阐明酶功能的结构/化学基础的模型。（本项目由分子生物化学和代谢生物化学联合资助。）