Preparation of bromo[1-14C]acetyl-coenzyme A as an affinity label for acetyl-coenzyme A binding sites.
Bromo[1-14C]acetyl-CoA has been prepared from CoASH and the N-hydroxysuccinimide ester of bromo[1-14C]acetic acid, and unlabeled bromoacetyl-CoA by reaction of CoASH with bromoacetyl bromide. The products were purified by high-pressure liquid chromatography. Purified bromoacetyl-CoA was characterized, and found to be a potent alkylating agent with a substantial stability in aqueous solution: it decomposed at 30 degrees C and pH 6.6 and 8.0 with halftimes of 3.3 and 2.5 h, respectively. The major breakdown products were CoASH and CoAS X CO X CH2 X SCoA. Bromo[1-14C]acetyl-CoA has been used to affinity label the acetyl-CoA binding site of 3-hydroxy-3-methylglutaryl-CoA synthase from ox liver. It was found to irreversibly inhibit the enzyme activity and bind covalently with a stoichiometry for complete inhibition of about 0.8 mol/mol enzyme dimer.
D000105:Acetyl Coenzyme A / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D000107:Acetylation; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000037:antagonists & inhibitors; D008099:Liver / Q000201:enzymology
D000345:Affinity Labels; D000105:Acetyl Coenzyme A; C033104:2-bromoacetyl coenzyme A; D006904:Hydroxymethylglutaryl-CoA Synthase