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Choline acetyltransferase: reversible inhibition by bromoacetyl coenzyme A and bromoacetylcholine.

Choline acetyltransferase (EC 2.3.1.6) catalyzes the following reversible reaction: acetyl coenzyme A + choline acetylcholine + coenzyme A. Bromoacetyl-CoA and bromoacetylcholine are potent inhibitors of the bovine brain enzyme. Although the enzyme is inactive immediately following Sephadex gel filtration to remove unreacted in- hibitor, enzyme activity returns to normal in 1 day at 0". When the enzyme is incubated with bromo( 14C)acetylcholine and the reaction mixture is passed through a Sephadex G-50 column, radiolabel is associated with the protein in the C holine acetyltransferase (EC 2.3.1.6) catalyzes the re- versible transfer of the acetyl group from acetyl-coenzyme A to choline. Previous studies are consistent with the hy- pothesis that an active-site sulfhydryl reacts with acetyl-co- enzyme A to form an acetyl-thioenzyme intermediate and coenzyme A (Roskoski, 1973). Choline then reacts with the acetyl-enzyme to form acetylcholine and the regenerated enzyme. Consonant with the notion of an active-site sulf- hydryl, thiol reagents inhibit choline acetyltransferase pre- pared from squid head ganglia (Reisberg, 1954), primate placenta (Schuberth, 1966), torpedo (Morris, 1967), and mammalian brain (Potter et al., 1968; Chao and Wolfgram, 1973). However, the bovine brain enzyme is protected from N-ethylmaleimide inactivation at high substrate concentra- tions during turnover, but becomes susceptable to inactiva- tion when the concentration of acetyl-coenzyme A (but not choline) is lowered (Roskoski, 1974). This suggests that the rate-limiting step during turnover is the reaction between choline with the acetyl-enzyme. Chase and Tubbs (1969) reported that pigeon breast muscle carnitine acetyltransferase (EC 2.3.1.7) is inhibited by bromo- acetyl-coA (in the presence of carnitine) or by bromoacetyl- carnitine (in the presence of CoA). They showed that a slowly dissociable S-carboxymethyl-CoA carnitine ester produced this inhibition. These workers subsequently showed that bromoacetylcarnitine irreversibly inhibited the enzyme by alkylation of a single histidine residue (Chase and Tubbs, 1970). Morris and Grewaal (1971) then reported that the human placental choline acetyltransferase is inhibited by bromoacetylcholine and analogous halogenated choline derivatives. The reversible nature of this inhibition led these eluent. When bromoacetyl(' Cjcholine is used, substantially less label is associated with the eluent protein. The bond be- tween the bromoacetyl group and the enzyme shows the characteristics of a thio ester. Kinetic studies indicate that bromoacetyl-CoA is a competitive inhibitor with respect to acetyl-CoA, and bromoacetylcholine is competitive with re- spect to acetylcholine. These experiments are consistent with the notion that the bromoacetyl derivatives acylate the active site sulfhydryl group and thereby inhibit the enzyme. filtration to remove the unreacted inhibitor, enzyme activity approaches control values within 1 day (t11~ = 6 hr). A bromo- acetyl-enzyme derivative was isolated by Sephadex gel filtra- tion using labeled bromo( 4C)acetylcholine and enzyme. The bromoacetyl-enzyme link shows the characteristics of a thio ester. The kinetic data show that bromoacetyl-CoA is a com- petitive inhibitor with respect to acetyl-coA and bromo- acetylcholine is competitive with respect to acetylcholine. These data are consistent with the hypothesis that the bromo- acetyl derivatives react with the active site -SH forming a thio ester and thereby inhibit the enzyme. The reactivation of the enzyme is apparently due to spontaneous hydrolysis of this bond.