Cohen_1982_J.Biol.Chem_257_14087

1-Bromopinacolone, BrPin, acts initially as a reversible competitive inhibitor for acetylcholinesterase, KI = 0.18 mM in hydrolysis of acetylcholine. Unlike bromoacetone, with time it acts as an irreversible covalent inhibitor. BrPin has a hydrolytic half-life of 30 h at the pH of incubation, 7.8. The enzyme-BrPin complex is 50% inactivated in 2 h. First order kinetics are observed; the rate constant is proportional to the concentration of complex. Retardation by cationic inhibitors of the inactivation is consistent with inactivation occurring as a result of binding of BrPin to the active site. Efficiency of irreversible inhibition by BrPin is essentially the same for hydrolysis of cationic and uncharged substrates, acetylcholine, 3,3-dimethylbutyl acetate, phenyl acetate, n-butyl acetate, and indophenyl acetate. In contrast, a cationic alkylating agent, N,N-dimethyl-2-phenylaziridinium ion, DPA, acts noncompetitively; it inactivates completely toward cationic, and partially toward uncharged substrates, and does so slightly more rapidly than BrPin, but less than would be commensurate with its greater intrinsic reactivity. Enzyme first treated with DPA is inactivated by BrPin toward hydrolysis of 3,3-dimethylbutyl acetate. It is proposed that BrPin, and not DPA, binds and reacts in, and may be a useful labeling agent for, the active site.