J. Biol. Chem., Vol. 257, Issue 23, 14087-14092, 12, 1982
1-Bromopinacolone, an active site-directed covalent inhibitor for acetylcholinesterase
SG Cohen, DL Lieberman, FB Hasan and JB Cohen
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.
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