Active site directed inactivation of rat mammary gland fatty acid synthase by 3-chloropropionyl coenzyme A

Abstract

3-Chloropropionyl coenzyme A (CoA) irreversibly inhibits rat mammary gland fatty acid synthase. Enzyme inactivation proceeds with first-order kinetics. NADPH (150 microM) as well as acetyl-CoA (500 microM) affords protection against inactivation, suggesting that the inhibitor is active site directed. In contrast, malonyl-CoA (500 microM) offers little protection. With chloro [1-14C]propionyl-CoA, stoichiometries of modification that approach one per enzyme protomer (240 kilodaltons) have been measured. When chloropropionyl-[3'-32P]CoA is used for inactivation, modification stoichiometries are less than 10% of the value observed in the 14C labeling experiments, suggesting that acylation of the enzyme occurs. Radioactivity remains associated with the 14C-labeled protein after performic acid oxidation, indicating that another linkage, in addition to the thio ester adduct, is formed during inactivation. Recovery of [( 14C]carboxyethyl)cysteine from digests of the inactivated enzyme indicates that alkylation of an active site cysteine occurs. The cysteamine sulfhydryl of the acyl carrier peptide is clearly not the site of modification. Loss of overall enzyme activity is tightly linked to decreases in the ketoacyl synthase partial reaction. This observation, coupled with the differential protection measured with acetyl-CoA and malonyl-CoA, suggests that the reagent modifies a residue at the active site involved in condensation. While inactivated enzyme shows good ketoacyl reductase activity when S-(acetoacetyl)-N-acetylcysteamine is used as a substrate, only poor activity for this partial reaction is measured when acetoacetyl-CoA is the substrate. This implies that the function of the acyl carrier peptide (ACP) is impaired during the inactivation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical events in chloropropionyl-coenzyme A inactivation of acyl-coenzyme A utilizing enzymes

“…Aliquots were withdrawn at the times indicated and assayed for enzyme activity by standard spectrophotometric procedures (Reed et al, 1975). * Values reported by Miziorko et al (1986).…”

“…Inactivation of Fatty Acid Synthase by S-Acrylyl-Nacetylcysteamine. Low levels of chloropropionyl-CoA efficiently inhibit fatty acid synthase (Miziorko et al, 1986).…”

“…Protein Chemistry Studies on Chloropropionyl-CoA-Modified Fatty Acid Synthase. The inactivation of fatty acid synthase by chloropropionyl-CoA has been explained by invoking carboxyethylation of a cysteinyl sulfhydryl in the condensing enzyme domain (Miziorko et al, 1986). The assignment was based on the observation that, after hydrolysis of enzyme that had been inactivated using radiolabeled inhibitor, the radiolabeled amino acid adduct comigrated with authentic (carboxyethyl)cysteine in two different thin-layer chromatographic systems.…”

“…The selectivity exhibited by this reagent facilitated isolation and sequence analysis of the modified site (Miziorko & Behnke, 1985b). Chloropropionyl-CoA also irreversibly modifies fatty acid synthase (Miziorko et al, 1986). A cysteine involved in the condensation reaction was implicated in this modification.…”

Medium-Chain Acyl-CoA Dehydrogenase- and Enoyl-CoA Hydratase-Dependent Bioactivation of 5,6-Dichloro-4-thia-5-hexenoyl-CoA

“…In the first, acryloyl-CoA generated in situ may react with a target within the active site of the dehydrogenase. A precedent is found in the generation of an acryloyl moiety from 3-chloropropionyl-CoA during the inactivation of fatty acid synthase and HMG-CoA synthase (Miziorko & Behnke, 1985;Miziorko et al, 1986). Neither the medium-chain enzyme (this work, not shown) nor the short-chain dehydrogenase (Shaw & Engel, 1984) is significantly inactivated by exogenously added acryloyl-CoA.…”