Adenylosuccinate Lyase: A Study of the Novel Intersubunit Active Sites

腺苷酸琥珀酸裂解酶通过将腺苷酸琥珀酸转化为AMP和延胡索酸来催化嘌呤生物合成中的关键步骤。 已经报道了在没有底物的情况下两种细菌酶的高分辨率结构。 在活性位点中发现了由两个不同亚基贡献的三个组氨酸。 然而，腺苷酸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酸琥珀酸裂解酶催化的主要化学和结构贡献，以及ASL缺乏症患者活性降低的分子基础。 具体目标解决三个问题：1。 腺苷酸琥珀酸裂解酶的哪些额外的氨基酸有助于催化和/或底物结合？ 这个问题是通过定点突变的氨基酸假定为在活性位点的B。枯草腺苷酸琥珀酸裂解酶 腺苷酸琥珀酸酯的稳定类似物，对酶裂解呈惰性，将被合成并用于制备晶体酶抑制剂复合物;它们也将被用于评估酶的底物结合。 2. 腺苷酸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，其中将成对的不同的无活性突变体混合并测试杂交四聚体中活性的恢复。 3. 腺苷酸琥珀酸裂解酶结构-功能关系的分子基础是什么？B。枯草腺苷酸琥珀酸裂解酶将被工程改造为具有与具有腺苷酸琥珀酸裂解酶缺陷的一些人变体中的氨基酸取代等同的氨基酸取代。 这些突变酶将进一步阐明酶功能的结构/化学基础的模型。(This该项目由分子生物化学和代谢生物化学计划共同支持。

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.)