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 D20 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 [1ICjacetylcoenzyme A were unsuccessful. A possible mechanism for the' action of choline acetyltransferase is proposed.Since acetylcholine plays an essential part in the permeability changes to cations in excitable membranes encountered during the conduction of the nervous impulse, a great deal of research has been centered on the enzymes responsible for the hydrolysis and the synthesis of this ester, acetylcholinesterase and choline acetyltransferase (ChA), respectively. ChA, the enzyme catalyzing the transfer of the acetyl group of acetylcoenzyme A to choline, has, since its discovery by Nachmansohn and Machado (1) in rabbit brain, been found in a wide variety of sources, including squid head ganglia (2, 3), rat brain (4), bovine brain (5), human placenta (6), and microorganisms (7). However, considerable difficulties were encountered in trying to obtain highly active, homogeneous material, ChA being a rather sluggish enzyme which tends to be unstable and which may exist in multiple forms (8-10). However, recently ChA with an activity of 4,000 smoles/hr per mg was isolated by Husain and Mautner from squid head ganglia (11).While ChA has been known for a long time, a great many questions regarding its mechanism of action remain unanswered. Evidence has been presented that the ChA catalyzed transacetylation from acetylcoenzyme A to choline involves an ordered or a Theorell-Chance mechanism (12-14) with acetylcoenzyme A as the leading substrate. Inhibition studies suggest the involvement of a thiol group in the functions of ChA (4, 15-17). It has also been proposed that imidazole may play a catalytic role in the functions of this enzyme (18,19 Corp.) and prostigmine, bovine serum albumin, ethylenediaminetetraacetate (EDTA), dithiothreitol, NaCl, NaPi, and 10 ;d of ChA (1.8 mg of protein per ml) in the usual amounts (23) with a final volume of 0.2 ml. The samples were incubated at 320 for 30 min; after chromatographic separation, the labeled acetylcholine formed was determined (23) with a Packard Tri-Carb liquid scintillation counter. All determinations were carried out in triplicate, as were controls lacking ChA.Inhibition Assays. Samples contained 50 mM choline, 50 mM inhibitor, 100 mM NaCl, as well as the other components noted above. All assays were carried out in triplicate as were blanks lacking ChA or inhibitor.

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.

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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.

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