Adenylosuccinate Lyase: Novel Intersubunit Active Sites

腺苷琥珀酸裂解酶：新型亚基间活性位点

• 批准号: 6414578
• 负责人: ROBERTA Fishman COLMAN
• 金额: $ 21.67万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6414578
• 关键词: Bacillus subtilis; active sites; adenosine monophosphate; arginosuccinate lyase deficiency; carbon nitrogen ligase; disease /disorder model; enzyme structure; molecular pathology; protein engineering; purine /pyrimidine metabolism; site directed mutagenesis; succinates; Bacillus subtilis; active sites; adenosine monophosphate; arginosuccinate lyase deficiency; carbon nitrogen ligase; disease /disorder model; enzyme structure; molecular pathology; protein engineering; purine /pyrimidine metabolism; site directed mutagenesis; succinates

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.

描述（由申请人提供）：腺苷核酸裂解酶催化2 嘌呤生物合成的独特但化学相关的步骤： 腺苷核酸到AMP +富马酸酯，裂解的裂解 5-氨基咪唑-4-（n-核酸羧酰胺）核糖核苷酸（SAICAR） 5-氨基咪唑-4-羧酰胺核糖核苷酸（AICAR） +富马酸盐。这 腺基核酸酶裂解酶（ASL）扮演的代谢作用的重要性是 由人类ASL缺乏症状的严重程度表示；这 疾病与智力低下，精神运动延迟，癫痫和 自闭症。 ASL被分离为相同亚基的四聚体。酶晶体 在没有底物或抑制剂的情况下，已经从3个细菌制备了 其中两个物种及其高分辨率结构已有报道。在 科尔曼博士的实验室，亲和力标签和定向的诱变 枯草芽孢杆菌ASL导致他的3识别（1168，H89和H141） 对于催化至关重要，其中2个可能充当一般酸/一般 反应过程中的基础，并由2个不同的亚基贡献。然而， 检查腺苷糖酸酯裂解酶的结构导致假设 在四酶四聚体中，3个亚基为4个中的每个亚基提供了氨基酸 活动地点。该项目的总体目标是了解专业 正常ASL和催化的化学和结构贡献 ASL缺乏患者活性降低的分子基础。 现在询问以下问题：ASL的哪些其他氨基酸 参与催化和/或底物结合，以及亚基提供的 这些残留物？这些问题将通过以网站为导向的诱变来解决 枯草芽孢杆菌ASL的氨基酸假定是在活性部位。目标的目标 将根据序列之间的保护选择诱变 来自28种的ASL和晶体结构的接近度 地点。突变酶将被表达，纯化为同质性并广泛 具有动力学，结合和生物物理测量的特征。 将进行互补实验，其中成对不同， 混合不活跃的突变体并测试杂种活性的恢复 四聚体。将评估双功能亲和力标签，以确定是否是否 亚基的交联。阐明ASL的分子基础 缺乏症，将用氨基酸构建枯草芽孢杆菌ASL的突变体 替换等于患有ASL缺乏的人类患者。这 突变酶将被纯化并广泛表征。目的是 使用这些突变酶作为模型来评估结构/化学基础 人类遗传缺陷。