INHIBITION OF 3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE AND CHOLESTEROL-BIOSYNTHESIS BY BETA-LACTONE INHIBITORS AND BINDING OF THESE INHIBITORS TO THE ENZYME

The beta-lactones L-659,699 {(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxetanyl]-3,5,7-trimethyl-2,4-undecadienoic acid} and its radioactive derivative H-3-L-668,411 (the 2,3-ditritiated methyl ester of L-659,699) inhibited a partially purified preparation of rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase with an IC50 of 0.1 muM. These compounds were also found to inhibit the incorporation of [C-14C]acetate into sterols in cultured Hep G2 cells with an IC50 of 3 muM. New kinetic evidence indicated that inhibition of the isolated enzyme was irreversible. In contrast, sterol biosynthesis in cultured Hep G2 cells was rapidly restored upon removal of the compound from the medium of inhibited cultures, suggesting reversibility of inhibition in the cells. Radioactivity was found to be associated with a single cytoplasmic protein by SDS/PAGE of the cytoplasm of Hep G2 cells after incubation of the cells with the inhibitor H-3-L-668,411. This protein was identified as cytoplasmic HMG-CoA synthase. Binding of the radioactive compound to the enzyme was decreased with time if the radioactive inhibitor was removed from the medium. Exposure of a gel containing the radioactive enzyme-inhibitor complex to neutral hydroxylamine also resulted in a loss of radioactivity from the gel. The purified rat liver enzyme reacted with the H-3-ligand to form a stable enzyme-inhibitor complex which could be isolated by h.p.l.c. Radioactivity was also subsequently lost from this complex when it was incubated with neutral hydroxylamine. Incorporation of [C-14]acetate into cholesterol in mouse liver was inhibited in a reversible manner after oral administration of the beta-lactone inhibitor. These studies, as well as the kinetic evidence presented, suggest that the beta-lactone inhibitors acylate HMG-CoA synthase in a reaction which appears to be irreversible in vitro, but is easily reversed in cultured cells and in animals.