STRUCTURE/FUNCTION OF CARBAMYL PHOSPHATE SYNTHETASE

氨甲酰磷酸合成酶的结构/功能

• 批准号: 2857080
• 负责人: CAROL J LUSTY
• 金额: $ 41.13万
• 依托单位: PUBLIC HEALTH RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2857080
• 关键词: Escherichia coli; X ray crystallography; active sites; adenosine triphosphate; allosteric site; carbamates; carbamoylphosphate synthase; conformation; crystallization; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; glutaminase; isozymes; mutant; phosphorylation; site directed mutagenesis; thioester

The long-term objective of this proposal is to define the structure- function relation in the E. coli enzyme, glutamine-dependent carbamyl phosphate synthetase (CPS). This enzyme with a unique mechanism of catalysis synthesizes a high energy precursor (carbamyl phosphate), required for pyrimidine biosynthesis in all living organisms. The protein has a complex mosaic structure with active sites related to the biotin-dependent carboxylases, and to a large family of enzymes that provide amide nitrogen for diverse biosynthetic reactions. At present, very little is known about these multifunctional enzymes and the manner in which their different active sites spatially and temporally interact during conversion of substrate to products. The specific aims are: (1) to continue the structure determination of E coli CPS by X-ray and multiwavelength anomalous diffraction analysis in a collaborative arrangement with Dr. Wayne Hendrickson's laboratory to solve the crystal structure; (2) to determine the structure of different catalytic and conformational forms of the protein; (3) to identify the functions of essential catalytic residues at each of the two ATP binding sites from hypotheses generated by the structure. Catalytic site mutants impaired in active site coupling are anticipated to provide additional mechanistic details of carbamate synthesis and phosphorylation; (4) to define residues essential to the function of the glutamine hydrolytic site, through site modification and kinetic analysis of thioester and product formation. New insights derived from the three-dimensional structure of CPS bear directly on the field of molecular medicine. Lesions in the human NH3-dependent enzyme either are lethal or lead to hyperammonemia, hepatic coma, and CNS damage. New directed enzyme therapies are continually being developed. Human pyrimidine-specific CPS is highly elevated in rapidly proliferating tumor tissue, and, therefore is a prime candidate for design of potent and selective inhibitors of the rapid tissue growth associated with cancer.

该提议的长期目标是定义结构 - 大肠杆菌酶，谷氨酰胺依赖性氨基甲酰基中的功能关系 磷酸合成酶（CPS）。 这种酶具有独特的机制 催化合成高能量前体（磷酸氨基甲酰基）， 在所有生物体中都需要嘧啶生物合成。 这 蛋白质具有复杂的镶嵌结构，具有与 生物素依赖性羧酸酶，以及大型酶家族 为各种生物合成反应提供酰胺氮。 现在， 关于这些多功能酶和方式知之甚少 其中其不同的活性位点在空间和时间上相互作用 在将基材转换为产品期间。 具体目的是：（1） 继续通过X射线和 在协作中的多波长异常衍射分析 与韦恩·亨德里克森（Wayne Hendrickson）博士的实验室进行安排，以解决水晶 结构; （2）确定不同催化和 蛋白质的构象形式； （3）确定功能 来自两个ATP结合位点的每个ATP结合位点的必需催化残基 结构产生的假设。 催化位点突变体受损 预计在主动站点中耦合会提供额外 氨基甲酸酯合成和磷酸化的机械细节； （4）至 定义对谷氨酰胺水解功能必不可少的残留物 站点，通过硫酯的现场修改和动力学分析和动力学分析 产品形成。 来自三维的新见解 CP的结构直接在分子医学领域。 人NH3依赖性酶的病变是致命的，要么是致死的 高症血症，肝昏迷和中枢神经系统损害。 新的定向酶 疗法正在不断开发。 人嘧啶特异性 CP在快速增殖的肿瘤组织中高度升高，并且 因此是设计有效和选择性的主要候选人 与癌症相关的快速组织生长的抑制剂。