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 hydroxymethylglutaryl-CoA (HMG-CoA) synthase is inactivated by oxobutylsulfoxyl-CoA in a time-dependent fashion. Protection against inactivation is afforded by the substrate, acetyl-CoA, suggesting that inactivation involves modification of the enzyme's active site. Pretreatment of HMG-CoA synthase with the inactivator blocks the enzyme's ability to form Michaelis and acetyl-S-enzyme intermediates, supporting the hypothesis that modification is active-site directed. Incubation of enzyme with oxobutylsulfoxyl-[32P]CoA followed by precipitation with trichloroacetic acid indicates 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 oxobutylsulfoxyl-CoA into a reactive species that modifies the protein. Treatment of inactivated enzyme with DTT or other mercaptans restores enzyme activity and reverses the covalent modification with release of CoASH. Oxobutylsulfoxyl-CoA inactivates beta-ketothiolase and HMG-CoA lyase in a process that is also reversed by DTT. These three enzymes all contain active site cysteines, suggesting that inactivation results from disulfide 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 disulfide linkage to protein.
Charlier
H A
HA
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226, USA.
Narasimhan
C
C
Miziorko
H M
HM
D000214:Acyl Coenzyme A / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology*; D000818:Animals; D002645:Chickens; D003545:Cysteine; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000037:antagonists & inhibitors*; D008956:Models, Chemical; D013329:Structure-Activity Relationship
C104507:3-oxobutylsulfonylcoenzyme A; D000214:Acyl Coenzyme A; D004791:Enzyme Inhibitors; D006904:Hydroxymethylglutaryl-CoA Synthase; D003545:Cysteine