Irreversible inhibition of fatty acid synthase from rat mammary gland with S-(4-bromo-2,3-dioxobutyl)-CoA. Effect on the partial reactions, protection by substrates and stoichiometry studies.
Fatty acid synthase from lactating rat mammary gland is rapidly and irreversibly inhibited by S-(4-bromo-2,3-dioxobutyl)-CoA. Of the seven partial reactions catalysed by the enzyme, the inhibition of the overall catalytic activity is closely paralleled only by inhibition of the beta-oxoacyl synthase (condensing) partial reaction. Three partial reactions. Beta-oxoacyl reductase, beta-hydroxyacyl dehydratase and enoyl reductase, are inhibited to a modest degree. The three partial reactions known to involve an acyl-CoA/CoA-binding site, acetyl acyltransferase, malonyl acyltransferase and palmitoyl thioesterase, are not inhibited by S-(4-bromo-2,3-dioxobutyl)-CoA. The modification process does not cause the enzyme to dissociate into catalytically incompetent monomers. Stoichiometric studies suggest that approx. 6 mol of reagent are incorporated per mol of totally inhibited enzyme (dimer). The formation of acylated enzyme from either acetyl-CoA or malonyl-CoA protects the enzyme equally well against S-(4-bromo-2,3-dioxobutyl)-CoA. Also, pretreatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid), a thiol-specific reagent reported to block essential thiol groups in the condensing partial reaction, protects against inhibition by the reagent. On the other hand, the presence of up to 770 microM-S-acetonyl-CoA or dethio-CoA does not protect the enzyme from irreversible inhibition. Together, the results suggest that the primary inhibitory process is a bimolecular reaction resulting in alkylation of essential thiol groups in the condensing partial reaction: this process does not require the obligatory formation of a Michaelis-Menten complex of enzyme and reagent before the alkylation reaction.
D000214:Acyl Coenzyme A / Q000494:pharmacology; D000215:Acylation; D000818:Animals; D001665:Binding Sites; D003065:Coenzyme A / Q000031:analogs & derivatives* / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors*; D005260:Female; D007700:Kinetics; D008321:Mammary Glands, Animal / Q000201:enzymology*; D051381:Rats
D000214:Acyl Coenzyme A; C017091:4-bromo-2,3-dioxobutyl-coenzyme A; D004228:Dithionitrobenzoic Acid; D064429:Fatty Acid Synthases; D003065:Coenzyme A