The Biochemistry of CAD

CAD 的生物化学

• 批准号: 9808562
• 负责人: Jeffrey Davidson
• 金额: $ 29.9万
• 依托单位: University of Kentucky Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9808562&HistoricalAwards=false
• 关键词: Biochemistry; CAD; Biochemistry; CAD

DavidsonPyrimidines are essential to normal cellular metabolism, proliferation and viability and to normal animal development. The research to be conducted is focused on the first three steps in the de novo synthesis of pyrimidines (i.e. the enzymes carbamyl phosphate synthetase [CPSase], aspartate transcarbamylase [ATCase], and dihydroorotase [DHOase]). In mammals, these three enzymes are found in a single translation product of ~240,000 daltons called CAD. Three specific aims will be pursued. The first is to learn how the carboxyl end of the DHOase domain contributes to enzymatic function when it is so different from its counterpart in E. coli. Specific site-directed substitutions made in the hamster DHOase will be analyzed for enzymatic kinetics both when the DHOase is expressed as a monoenzymatic protein and as a domain in CAD. The second aim is to identify those residues in the allosteric domain of the CPSase that bind the negative effector, UTP, and the positive effector, PRPP. CAD carrying substitutions in the allosteric domain will be assayed in vitro for activity in the presence or absence of UTP and PRPP and will be introduced into Drosophila in order to see how alterations in allosteric regulation affect fly development and pyrimidine levels. The third aim is to examine the pattern of regulated CAD expression in mouse embryos and in adult mouse organs. Tissue slices will be hybridized with 35S-labeled CAD antisense RNA to reveal those cells expressing CAD RNA and by inference the de novo pyrimidine biosynthetic pathway. The results of this research will provide new data on the structure and function of an essential multienzymatic protein, on the importance of regulating the first step in a biosynthetic pathway and on when and where CAD is expressed in animal tissues.

Davidsonpyrimidines对于正常的细胞代谢，增殖和生存力以及正常动物发育至关重要。进行的研究集中于嘧啶从头合成的前三个步骤（即，氨基氨基磷酸酶合成酶[CPSase]，天冬氨酸经钙化酶[actcase]和二羟基drootorotose [cpsassase]和二氢核苷酸[Dhoase]）。在哺乳动物中，这三种酶是在约240,000个Daltons的单个翻译产品中发现的，称为CAD。将追求三个具体目标。首先是学习Dhoase结构域的羧基端如何在与大肠杆菌中的对应物如此不同时促进酶促功能。当Dhoase表示为单酶蛋白和CAD中的域时，将分析仓鼠dhoase中的特定位置定向的取代。第二个目的是识别CPSase的变构域中的那些残基，该残基结合负效应子UTP和阳性效应子PRPP。在存在或不存在UTP和PRPP的情况下，将在体外测定变构域中的CAD携带取代，并将被引入果蝇中，以了解变构调节的变化如何影响苍蝇的发育和嘧啶水平。第三个目的是检查小鼠胚胎和成年小鼠器官中调节的CAD表达的模式。组织切片将与35S标记的CAD反义RNA杂交，以揭示表达CAD RNA的细胞，并通过推断从头嘧啶生物合成途径。这项研究的结果将提供有关必需多酶蛋白的结构和功能的新数据，该数据关于调节生物合成途径的第一步以及在动物组织中表达CAD的何时何地的重要性。