INACTIVATION OF 3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE AND OTHER ACYL-COA-UTILIZING ENZYMES BY 3-OXOBUTYLSULFOXYL-COA

3-Oxobutylsulfoxyl-COA has been produced by oxidation of S-3-oxobutyl- CoA, the thioether analog of acetoacetyl-CoA. Avian hydroxymethylgluta ryl-CoA (HMG-CoA) synthase is inactivated by oxobutylsulfoxyl-CoA in a time-dependent fashion. Protection against inactivation is afforded b y the substrate, acetyl-CoA, suggesting that inactivation involves mod ification of the enzyme's active site. Pretreatment of HMG-CoA synthas e with the inactivator blocks the enzyme's ability to form Michaelis a nd acetyl-S-enzyme intermediates, supporting the hypothesis that modif ication is active-site directed. Incubation of enzyme with oxobutylsul foxyl-[P-32]CoA followed by precipitation with trichloroacetic acid in dicates that inactivation correlates with stoichiometric formation of a covalent adduct between enzyme and a portion of the inactivator that includes the CoA nucleotide. The observation of reagent partitioning suggests that HMG-CoA synthase catalyzes conversion of oxobutylsulfoxy l-CoA into a reactive species that modifies the protein. Treatment of inactivated enzyme with DTT or other mercaptans restores enzyme activi ty and reverses the covalent modification with release of CoASH. Oxobu tylsulfoxyl-CoA inactivates beta-ketothiolase and HMG-CoA lyase in a p rocess that is also reversed by DTT. These three enzymes all contain a ctive site cysteines, suggesting that inactivation results from disulf ide formation between a cysteine and the CoA moiety of the inhibitor. The data are consistent with the hypothesis that-enzymatic cleavage of oxobutylsulfoxyl-CoA results in the transient formation of a sulfenic acid derivative of CoA which subsequently reacts to form a stable dis ulfide linkage to protein.