Adenylosuccinate Lyase: Novel Intersubunit Active Sites

说明（由申请人提供）：腺苷酸琥珀酸裂解酶催化2 嘌呤生物合成中不同但化学相关的步骤： 腺苷酸琥珀酸酯转化为AMP+富马酸酯， 5-氨基咪唑-4-（N-琥珀酰甲酰胺）核糖核苷酸（SAICAR）， 5-氨基咪唑-4-甲酰胺核苷酸（AICAR）+富马酸盐。的 腺苷酸琥珀酸裂解酶（ASL）所起的代谢作用的重要性是 由人类ASL缺乏症症状的严重程度指示; 疾病与智力迟钝、精神发育迟滞、癫痫和 自闭症ASL被分离为相同亚基的四聚体。酶晶体 在不存在底物或抑制剂的情况下，从3种细菌制备了 已报道了其中两种的物种和高分辨率结构。在 科尔曼博士的实验室，亲和标记和定点诱变的 枯草芽孢杆菌ASL导致鉴定3个His（1168、H89和H141） 作为催化剂的关键，其中2种可能用作一般酸/一般酸 在反应过程中的碱基，并由2个不同的亚基贡献。然而，在这方面， 对腺苷酸琥珀酸裂解酶结构的研究导致了以下假设 在酶四聚体中，3个亚基为4个亚基中的每一个提供氨基酸， 活性位点。这个项目的总体目标是了解主要的 化学和结构的贡献，催化正常ASL和 ASL缺乏患者活动减少的分子基础。 以下问题正在被提出：ASL中的哪些额外氨基酸 参与催化和/或底物结合，并且哪些亚基提供 这些残留物？这些问题将通过定点诱变来解决， B的氨基酸。枯草杆菌ASL假定是在活性位点。目标 诱变将基于序列之间的保守性来选择 ASL从28种和接近的晶体结构的活性 绝佳的价钱突变酶将被表达、纯化至均一并广泛地 通过动力学、结合和生物物理测量表征。 将进行互补实验， 将失活突变体混合并测试杂种中活性的恢复 四聚体。将评价双功能亲和标记物以确定是否 发生亚单元的交联。阐明ASL的分子基础 缺陷，B的突变体。将用氨基酸构建枯草杆菌ASL 与具有ASL缺陷的人类患者中的那些等同的置换。的 突变酶将被纯化和广泛表征。目的是 使用这些突变酶作为模型来评估结构/化学基础， 人类基因缺陷的证据

DESCRIPTION (provided by applicant): Adenylosuccinate lyase catalyzes 2 distinct but chemically related steps in purine biosynthesis: the conversion of adenylosuccinate to AMP + fumarate, and the cleavage of 5-aminoimidazole-4-(N-succinylocarboxamide)ribonucleotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) + fumarate. The importance of the metabolic role played by adenylosuccinate lyase (ASL) is indicated by the severity of the symptoms of ASL deficiency in humans; the disease is associated with mental retardation, psychomotor delay, epilepsy and autism. ASL is isolated as a tetramer of identical subunits. Crystals of enzyme in the absence of substrate or inhibitor have been prepared from 3 bacterial species and high-resolution structures have been reported for two of them. In Dr. Colman's laboratory, affinity labeling and site-directed mutagenesis of the Bacillus subtilis ASL led to the identification of 3 His (1168, H89 and H141) as critical for catalysis, 2 of which likely function as general acid/general base during the reaction and are contributed by 2 different subunits. However, examination of the structure of adenylosuccinate lyase leads to the hypothesis that in an enzyme tetramer, 3 subunits provide amino acids to each of the 4 active sites. The overall goal of this project is to understand the major chemical and structural contributions to catalysis by normal ASL and the molecular basis for decreased activity in patients with ASL deficiency. Following questions are being now asked: which additional amino acids of ASL participate in catalysis and/or substrate binding, and which subunits provide these residues? These issues will be approached by site-directed mutagenesis of amino acids of B. subtilis ASL postulated to be at the active site. Targets of mutagenesis will be selected on the basis of conservation among the sequences of ASL from 28 species and proximity in the crystal structures to the active site. Mutant enzymes will be expressed, purified to homogeneity and extensively characterized by kinetics, binding and biophysical measurements. Complementation experiments will be conducted in which pairs of different, inactive mutants are mixed and tested for restoration of activity in hybrid tetramers. Bifunctional affinity labels will be evaluated to ascertain whether crosslinking of subunits occurs. To elucidate the molecular basis of ASL deficiency, mutants of B. subtilis ASL will be constructed with amino acid substitutions equivalent to those in human patients with ASL deficiency. The mutant enzymes will be purified and extensively characterized. The intention is to use these mutant enzymes as models to evaluate the structural/chemical basis of a human genetic defect.