Incubation of 3-chloropropionyl-CoA with 3-hydroxy-3-methylglutaryl-CoA synthase results in exchange of the C2 proton with solvent as inactivation of enzyme proceeds. This enzyme is also inhibited by S-acrylyl-N-acetylcysteamine; the limiting rate constant for inactivation by the acrylyl derivative (0.36 min-1) slightly exceeds the value measured for chloropropionyl-CoA (0.31 min-1). These observations support the intermediacy of acrylyl-CoA in the chloropropionyl-CoA-dependent inactivation of hydroxymethylglutaryl-CoA synthase. Inhibition of fatty acid synthase by chloropropionyl-CoA is primarily due to alkylation of a reactive cysteine, although secondary reaction with the enzyme's pantetheinyl sulfhydryl occurs. Modification of fatty acid synthase by S-acrylyl-N-acetylcysteamine occurs at a limiting rate (1.8 min-1) that is comparable to that estimated for chloropropionyl-CoA-dependent inactivation. However, this enzyme lacks the ability to deprotonate C2 of an acyl group such as the chloropropionyl moiety. Since such a step would be required to generate an acrylyl group from chloropropionyl-S-enzyme, it is likely that a typical affinity labeling process accounts for inactivation of fatty acid synthase by chloropropionyl-CoA. HMG-CoA lyase is also inhibited by S-acrylyl-N-acetylcysteamine. In contrast to the ability of this reagent to serve as a mechanism-based inhibitor of hydroxymethylglutaryl-CoA synthase and an affinity label of fatty acid synthase, it acts as a group-specific reagent in modifying HMG-CoA lyase (kappa 2 = 86.7 M-1 min-1).