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.
