Investigations on the covalent modification of thiolase by potential hypolipidemic agents

This work was designed to test several classes of inhibitors on liver cytoplasmic acetoacetyl-CoA thiolase (EC 2.3.19) and heart mitochondrial p-ketoacyl CoA thiolase (EC 2.3.1.16). These inhibitors are based on a new reactive group for alkylation studies, the thiosulfonate group, which readily modifies enzyme sulphydryl groups. The newly synthesised compounds were bifunctional thiolsulfonates of the general structure, CH SO S(CH2 ) SSO2CH3 (n=2 - 12) and CH (CH ) COCH SSO CH . Methyl methanethiosulfonate, (MMTS) injected thiolase in a time dependant reaction and this was protected by acetoacetyl C0A and acetyl CoA. The thiomethyl group could be removed by reduction. This active site directed thiomethylation allowed the study secondary amino modification (succinic, phthalic, and citraconic anhydrides) which also inactivted thiolase in an active site directed manner. When the enzyme was double modified MMTS and citraconic anhydride, the thiomethyl group could be reduced by excess thiols and the amino group could be unblocked by exposure to acidic pH's. The reduction of thethiomethyl-citraconyl thiolase left the enzyme inactive, I whereas exposure to pH 5 followed by reduction led to the formation of an active enzyme. These results are interpreted as demonstrating a role for an amino group of the enzyme active site. A catalytic mechanism is proposed for the enzyme which incorporates this group.In the second part, the compounds were tested aspotential hypolipidemic agents. This was correlated with their inhibitory effects on the cytoplasmic isoenzyme of I thiolase in vitro. In vitro, the compounds inactivated the enzyme in a psuedo first order kinetic pattern in the order of CH (CH? )' COCH'Cl> n=12> n=10> n=8> n=6>CH (CH )1 SS02Cn > n=5> n=2. In vivo when drugs were injected into groups of newly weaned rats fed on a highcarbohydrate or high fat diet, the compounds had a significant hypocholestrolemic effect and a profound hypotriglyceridemic action. Their hypocholesterolemic was in the order of n=12> CH 3(CH2)14000H2SSO2CH3>CH (CH ) COCHCl> n= 8> n=6, and their hypotriglyceridemic effect was nJ> CH (CH ) COCH SSO CH > CH (CH ) COCH C1> n=12> n=10. Their hypolipidemic action resulted in a mild hypoglyceamia, and their growth rate, protein catabolism (BUN, blood total proteins) was normal. The injections of the drugs did not result in any deposition of atherosclerotic lesions in the aotras. When thiolase activity in isolated liver homogenates was measures after injections of drugs in vivo, the enzyme was inactivated by 80%. This inhibition of enzyme activity does not seem enough to account for the hypolipidemic action in vivo since these effects did not correlate with inhibition of the isolated enzyme. I conclude that the drugs must be exerting their effect by influencing additional unidentified thiol dependant processes.