SQUALENE SYNTHASE--STRUCTURE, FUNCTION AND INHIBITION

这项建议的主要目的是研究第一种酶的特异性， 在肝脏胆固醇生物合成途径中形成甾醇， 角鲨烯合酶，以了解参与的具体 氨基酸残基的蛋白质在不同阶段的详细 催化过程此外，还将设计合成抑制剂， 这将有助于我们理解催化过程， 可能导致胆固醇降低剂的发展。这些 目标现在是可以实现的，因为我们最近分离，纯化， 克隆并表达大鼠肝角鲨烯合成酶（RSS）。计算机 还进行了RSS二级结构的建模，导致 鉴定涉及的三个蛋白质序列（A、B和C部分） 在催化部位。 研究了特定残留物在不同阶段的参与， 所述催化将包括通过使用 定点突变技术修饰的cDNA， RSS和突变酶在原核细胞中的表达。的 将研究突变酶的催化性质， 因此，修饰残基的贡献和参与 将被评估。 基于蛋白质的结构信息，在理论上考虑， 关于催化过程和关于初步成功地编写 合成磺基甜菜碱两性离子抑制剂，新的化合物将被 制备，并将检查它们作为RSS抑制剂的效果。 这种研究结构/功能关系的双向方法， 涉及结合DNA重组技术进行修饰 酶的催化位点的设计，以及特定的 另一方面，抑制剂，应该大大增加我们对 催化和调节这种重要的酶， 胆固醇生物合成途径

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.