Structure-Function Analysis of Enzymes

描述：（申请人的描述）基底沟道的概念是 最初提出来解释反应性中间体被 在代谢途径中从一种蛋白质转移到另一种蛋白质，或者从 一个酶的活性位点与另一个酶的活性位点相连。许多人认为， 现象作为一个关键的方法，为调节代谢途径内 活细胞。该提案的目的是探索，在一个结构上， 在此基础上，以下已知的酶系统的作用机制， 利用底物通道：氨甲酰磷酸合成酶，天冬酰胺 合成酶和乙酰辅酶A羧化酶。前两种酶利用底物 在单个多肽链内的活性位点之间形成通道，而在 后一种蛋白质，事件发生在位于不同的活性位点之间， 多肽链。选择这些系统进行研究不仅是因为它们 在它们的反应机制中使用底物沟道，而且还因为 生物学的重要性。氨甲酰磷酸合成酶参与了 嘧啶核苷酸的从头合成， 增殖这种酶在精氨酸的生物合成中也起着关键作用， 以及大多数陆生脊椎动物的尿素循环。天冬酰胺合成酶 催化天冬氨酸向天冬酰胺的ATP依赖性转化， 用于治疗急性淋巴细胞白血病的化疗方案中， 白血病最后，乙酰辅酶A羧化酶催化第一个承诺， 脂肪酸合成的关键步骤。对于这些调查， 定点诱变实验和X射线晶体学的组合 将进行分析，以便更全面地描述 这些蛋白质的三维结构，它们的活性位点， 几何形状，催化机理，以及这些参数与 衬底沟道效应

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.