Ha. Charlier et al., INACTIVATION OF 3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE AND OTHER ACYL-COA-UTILIZING ENZYMES BY 3-OXOBUTYLSULFOXYL-COA, Biochemistry, 36(6), 1997, pp. 1551-1558
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.