SQUALENE SYNTHASE--STRUCTURE, FUNCTION AND INHIBITION

角鲨烯合成酶——结构、功能和抑制

• 批准号: 2226875
• 负责人: ISHAIAHU SHECHTER
• 金额: $ 32.92万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2226875
• 关键词: antihypercholesterolemic agent; complementary DNA; enzyme inhibitors; enzyme mechanism; gene expression; molecular site; protein structure function; recombinant proteins; site directed mutagenesis; squalene; steroid biosynthesis

The main objective of this proposal is to study the first enzyme specific to sterol formation in the cholesterol biosynthetic pathway in the liver, squalene synthase, in order to understand the involvement of specific amino acid residues of the protein in the different stages of the detailed catalytic process. In addition, synthetic inhibitors will be designed and prepared which will aid in our understanding of the catalytic process and perhaps result in the development of cholesterol lowering agents. These objectives are now attainable since we have recently isolated, purified, cloned and expressed the rat hepatic squalene synthase (RSS). Computer modeling of RSS secondary structure has also been conducted, leading to the identification of three protein sequences (Sections A, B & C) involved in the catalytic site. The study of the involvement of specific residues in different stages of the catalysis will involve modification of these residues by employing site directed mutagenesis technology in the modification of the cDNA for RSS and expression of the mutated enzyme in procaryotic cells. The catalytic properties of the mutated enzyme will be studied and, accordingly, the contribution and involvement of the modified residues will be assessed. Based on protein structural information, on theoretical considerations of the catalytic process and on preliminary success in the preparation of synthetic sulfobetaine zwitterionic inhibitors, new compounds will be prepared and their effect as inhibitors of RSS will be examined. This two way approach to a study of structure/function relationship, involving a combination of DNA recombinant technology for the modification of the enzyme's catalytic site on one hand, and the design of specific inhibitors on the other, should vastly increase our understanding of the catalysis and perhaps regulation of this important enzyme in the cholesterol biosynthetic pathway.

该提案的主要目的是研究第一个酶特异性 在肝脏中的胆固醇生物合成途径中形成固醇， 为了了解特定的参与，鳞状合酶 蛋白质的氨基酸残基在详细的不同阶段 催化过程。此外，将设计合成抑制剂，并 准备的将有助于我们理解催化过程和 也许会导致降低胆固醇剂的发展。这些 现在可以实现目标，因为我们最近隔离，纯化， 克隆并表达大鼠肝棘磷酸合酶（RSS）。电脑 还进行了RSS二级结构的建模，导致 涉及三个蛋白质序列（A，B＆C）的鉴定 在催化部位。 研究特定残基在不同阶段的参与的研究 催化将涉及通过采用这些残基的修改 站点定向诱变技术，用于修饰cDNA RSS和突变细胞中突变酶的表达。这 将研究突变酶的催化特性，并 因此，修饰残基的贡献和参与 将被评估。 基于蛋白质结构信息，理论上的考虑 催化过程和关于制备的初步成功 合成硫素兹链离子抑制剂，新化合物将是 将检查制备及其作为RSS抑制剂的作用。 这种研究结构/功能关系的两种方法， 涉及DNA重组技术进行修饰的组合 一方面的酶催化位点的设计 另一方面的抑制剂应大大增加我们对 催化，也许调节这种重要酶 胆固醇生物合成途径。