Adenylosuccinate Lyase: Novel Intersubunit Active Sites

描述(申请人提供)：腺基琥珀酸裂解酶催化2 嘌呤生物合成中不同但化学相关的步骤： 腺苷琥珀酸到富马酸AMP，以及 赛卡(5-aminoimidazole-4-(N-succinylocarboxamide)ribonucleotide)至 5-氨基咪唑-4-甲酰胺核糖核苷酸(AICAR)富马酸盐。这个 腺苷琥珀酸裂解酶(ASL)代谢作用的重要性在于 由人类ASL缺乏症状的严重性来表示； 疾病与智力低下、精神运动迟缓、癫痫和 自闭症。ASL是作为相同亚基的四聚体分离出来的。酶的结晶 在没有底物或抑制剂的情况下，从3个细菌制备 其中两个化合物的物种和高分辨率结构已被报道。在……里面 Colman博士的实验室、亲和标记和定点突变 枯草芽孢杆菌ASL鉴定出3株His(1168、H89和H141) 作为催化的关键，其中2个可能作为总酸/总酸起作用 反应中的碱基，由两个不同的亚基组成。然而， 对腺苷琥珀酸裂解酶结构的研究导致了这一假说 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 活动站点。本项目的总体目标是了解 正常ASL和ASL对催化的化学和结构贡献 ASL缺乏症患者活性降低的分子基础。 现在提出了以下问题：ASL的哪些额外氨基酸 参与催化和/或底物结合，以及哪些亚基提供 这些残留物？这些问题将通过定点突变来解决。 推测枯草杆菌ASL的氨基酸位于活性部位。目标 突变将在序列保守的基础上进行选择 ASL的28个物种及其晶体结构与活性的亲和性 地点。突变的酶将被表达、纯化到同质性和广泛性 以动力学、结合和生物物理测量为特征。 将进行互补性实验，其中不同的对， 不活跃的突变体混合在一起，并进行测试，以恢复杂交的活性 四聚体。将对双功能亲和标签进行评估，以确定 亚基发生交联化。阐明ASL的分子基础 枯草杆菌ASL缺陷型突变株将用氨基酸构建 等同于人类ASL缺乏症患者的替换。这个 突变的酶将被提纯并进行广泛的鉴定。其意图是 用这些突变的酶作为模型来评估结构/化学基础 人类的基因缺陷。

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.