REGULATION AND MECHANISM OF PRPP SYNTHETASE

建议研究催化的代谢调控和机制 磷酸核糖焦磷酸（phosphoribosylpyrophosphate，PRPP） 来自肠细菌鼠伤寒沙门氏菌（Salmonella typhimurium）的合成酶。 这种酶 特别令人感兴趣，因为（a）PRPP是 通用的，高度分支的途径，导致所有核苷酸，两个氨基 （B）该酶催化一种不寻常的 焦磷酰基转移反应，和（c）PRPP代谢是已知的， 在许多遗传性代谢疾病中发挥重要作用， 贫血或高尿酸血症和痛风。 建议克隆 来自S.鼠伤寒沙门氏菌，并确定 它的核苷酸序列。 这将提供 酶;活性位点氨基酸残基将被定位在序列中， 用活性定点试剂和肽进行化学修饰 映射. 这些结果，结合以前和提出的机制 研究，应该提供一个更精细的图片酶催化PRPP 合成酶 PRPP合成酶基因的转录调控 将通过对相邻对照区进行测序和研究 使用在重组质粒上分离的DNA进行体外转录。 的 将探讨影响PRPP合成酶活性的第二个基因的功能 通过类似的技术。

It is proposed to study the metabolic regulation and mechanism of catalysis of the essential biosynthetic enzyme phosphoribosylpyrophosphate (PRPP) synthetase from the enteric bacterium Salmonella typhimurium. This enzyme is of particular interest because (a) PRPP is the first step in a universal, highly branched pathway leading to all nucleotides, two amino acids, and two coenzymes; (b) the enzyme catalyzes an unusual pyrophosphoryl group transfer reaction, and (c) PRPP metabolism is known to play an important role in a number of inherited metabolic diseases leading to anemia or hyperuricemia and gout. It is proposed to clone the structural gene for PRPP synthetase from S. typhimurium and to determine its nucleotide sequence. This will provide the primary structure of the enzyme; active site amino acid residues will be located in the sequence by chemical modification with active site-directed reagents and peptide mapping. These results, combined with previous and proposed mechanistic studies, should provide a much refined picture of enzymic catalysis by PRPP synthetase. The regulation of transcription from the PRPP synthetase gene will be studied by sequencing adjacent control regions and by studying transcription in vitro using DNA isolated on recombinant plasmids. The function of a second gene affecting PRPP synthetase activity will be probed by similar techniques.