ENZYME-INHIBITION DURING THE CONVERSION OF SQUALENE TO CHOLESTEROL

Two separate enzymatic assays were developed in order to test the sele ctivity of inhibitors in cholesterol biosynthesis. One assay detects i nhibition of Delta(5,7)-sterol Delta(7)-reductase, the enzyme involved in the conversion of 7-dehydrocholesterol to cholesterol. Delta(5,7)- Sterol Delta(7)-reductase was inhibited by both RPR 101821, a protonat ed cyclohexylamine, and BM 15.766, a piperazine derivative, with IC50 values of 1 mu M. The second assay detects accumulation of any of five intermediates (squalene oxide, squalene dioxide, lanosterol, desmoste rol, and 7-dehydrocholesterol) upon inhibition of enzymes catalyzing r eactions in the conversion of squalene to cholesterol. In this assay, inhibition data were most accurate when control assays exhibited a con version of squalene to cholesterol in the order of 50%. The time requi red to attain 50% conversion of squalene to cholesterol was 6 h. Given a high inhibitor to substrate concentration ratio and the possible va lues of K-i, k(on), and k(off) for the reaction between enzymes and in hibitor to form enzyme-inhibitor complexes, it was predicted that in t he presence of inhibitors, intermediate accumulation could still be ob served after 6 h incubation. The experimental results were in agreemen t with this prediction.