Choline Acetyltransferase–evidence for Acetyl Transfer by a Histidine Residue

Choline acetyltransferase from bovine brain has been extensively purified to a specific activity of 2.5 μmol ACh/min mg protein. Attempts to isolate an acetyl enzyme intermediate after incubation of the enzyme with [1‐ 14 C]acetyl‐CoA were unsuccessful. Such an intermediate could only be isolated using a 30‐fold less purified enzyme preparation. The protein, binding 14 C in this preparation, did not correspond to choline acetyltransferase as shown by disc‐electrophoresis. The highly purified enzyme could, however, be labelled when choline acetyltransferase was immobilized on a mercuribenzoate sepharose gel and incubated with [1‐ 14 C]acetyl‐CoA. Subsequently, the immobilized labelled enzyme or the labelled enzyme which had been released by cysteine from the gel. formed ACh after incubation with choline. The labelling and the following formation of [ 14 C]ACh was pH dependent. Masking htstidine residues of the enzyme with diethylpyrocarbonate almost abolished the labelling of the immobilized enzyme and completely abolished the formation of [ 14 C]ACh. Enzyme inhibited with 5.5′‐dithiobis(2‐nitrobenzoate) was partially reactivated when the thionitrobenzoatederivative was cleaved by KCN treatment to a thiocyanatederivalive. A reaction mechanism for ChAT is proposed based on the present data.