Structure-Function Analysis of Enzymes

描述：(申请人描述)衬底沟道的概念是 最初提出的目的是解释活性中间体是如何 在代谢途径中从一种蛋白质转移到另一种蛋白质或从 在单个酶中从一个活性部位到另一个活性部位。许多人认为 现象作为调节体内代谢途径的关键方法 活细胞。这项提案的目标是探索一种结构性的 在此基础上，已知下列酶系统的作用机制 使用底物通道：氨基甲酰磷酸合成酶，天冬酰胺 合成酶和乙酰辅酶A羧基酶。前两种酶利用底物 在单一多肽链中的活性部位之间的通道 后一种蛋白质，该事件发生在位于不同 多肽链。选择这些系统进行研究不仅是因为它们 在他们的反应机理中使用底物沟道，也是因为 它们在生物学上的重要性。氨基甲酰磷酸合成酶参与了 对细胞至关重要的嘧啶核苷酸的从头合成 扩散。该酶在精氨酸的生物合成中也起着关键作用。 大多数陆地脊椎动物的尿素循环。天冬酰胺合成酶 催化三磷酸腺苷依赖的天冬氨酸转化为天冬酰胺 用于急性淋巴母细胞瘤的化疗方案 白血病。最后，乙酰辅酶A羧基酶催化第一个承诺的和 脂肪酸合成中绝对关键的一步。在这些调查中， 定点诱变实验与X射线结晶学相结合 将采用分析，以便更全面地描述 这些蛋白质的三维结构，它们的活性部位 几何构型、催化机理以及这些参数之间的关系 衬底沟道。

DESCRIPTION: (Applicant's Description) The concept of substrate channeling was originally put forth to explain the manner in which reactive intermediates are transferred from one protein to another in a metabolic pathway or shuttled from one active site to another within a single enzyme. Many have regarded the phenomenon as a critical method for the regulation of metabolic pathways within the living cell. The goal of this proposal is to explore, on a structural basis, the mechanisms of actions of the following enzymatic systems known to employ substrate channeling: carbamoyl phosphate synthetase, asparagine synthetase, and acetyl CoA carboxylase. The first two enzymes utilize substrate channeling between active sites within a single polypeptide chain while in the latter protein, the event occurs between active sites positioned on different polypeptide chains. These systems were selected for study not only because they use substrate channeling in their reaction mechanisms, but also because of their biological importance. Carbamoyl phosphate synthetase is involved in the de novo synthesis of pyrimidine nucleotides which are critical for cell proliferation. The enzyme also plays a key role in both arginine biosynthesis and the urea cycle in most terrestrial vertebrates. Asparagine synthetase catalyzes the ATP-dependent conversion of aspartic acid to asparagine and has been used in chemotherapeutic protocols for treating acute lymphoblastic leukemia. Finally, acetyl CoA carboxylase catalyzes the first committed and absolutely critical step in fatty acid synthesis. For these investigations, a combination of site-directed mutagenesis experiments and x-ray crystallographic analyses will be employed in order to more fully characterize the three-dimensional architectures of these proteins, their active site geometries, catalytic mechanismd, and the relationships of these parameters to substrate channeling.