RESUMEN
The interaction of a series of aromatic dyes with the coenzyme A binding site of choline acetyltransferase was studied. Several of the dyes were very potent inhibitors of the enzyme. With few exceptions, inhibition was competitive with respect to acetylcoenzyme A and noncompetitive with respect to choline. It appears likely that inhibition by dyes such as Reactive Blue 2 (Cibacron Blue F3GA) or Congo Red, as in the case of coenzyme A interactions, involves hydrophobic bonding, as well as a coulombic interaction with an arginine residue.
Asunto(s)
Colina O-Acetiltransferasa/antagonistas & inhibidores , Coenzima A/metabolismo , Colorantes/farmacología , Animales , Sitios de Unión/efectos de los fármacos , Colina O-Acetiltransferasa/metabolismo , Decapodiformes , Ganglios/enzimología , Técnicas In Vitro , CinéticaRESUMEN
The finding that methyl methanethiolsulfonate appears to inhibit choline acetyltransferase from squid ganglia not by reacting with a thiol group of the enzyme but by reacting with the thiol group of coenzyme A to form a competitive inhibitor of acetyl coenzyme A led to the synthesis of the ethyl, propyl, and 3-carboxy-4-nitrophenyl disulfides of CoA. The methyl disulfide of 1,N6-etheno-C0A, a fluorescent ligand, was also prepared. All the disulfides are powerful inhibitors of ChA, their Ki values being very similar. The Km values for acetylpropionyl-, and butyryl-CoA were also found to be similar; however, modification of the acyl group alter the Km values for choline. CoA, and dethia-CoA, showed similar abilities to be bound to ChA; however, the 3'-phospho groups of acetyl CoA and CoA appear to be of importance in interacting with the enzyme. 8-Anilino-1-naphthalenesulfonate is a competitive inhibitor of acetyl-CoA binding.
Asunto(s)
Acetiltransferasas/metabolismo , Colina O-Acetiltransferasa/metabolismo , Coenzima A/análogos & derivados , Animales , Colina O-Acetiltransferasa/antagonistas & inhibidores , Coenzima A/metabolismo , Decapodiformes , Ganglios , Cinética , Relación Estructura-ActividadRESUMEN
The substrate specificity of choline acetyltransferase (EC 2.3.1.6) isolated from squid ganglia was investigated. The enzyme catalyzed the acetylation of choline and aminocholine but not of homocholine. In D(2)O solution there was considerable slowing of the transacetylation reaction. Photo-oxidation in the presence of methylene blue or rose bengal rapidly inactivated the enzyme, suggesting involvement of a histidine residue in the catalytic site. It seems likely that general-base catalysis by imidazole enhances the ability of enzyme-bound choline (or ammoniumcholine) to react with a thiolester group. Attempts to isolate an acetylthio-enzyme intermediate after incubation with [(14)C]acetylcoenzyme A were unsuccessful. A possible mechanism for the action of choline acetyltransferase is proposed.