Add time:07/15/2019         Source:sciencedirect.com

1-Bromo-2-[14C]pinacolone, (CH3)3C14COCH2Br ([14C]BrPin), was prepared from [1-14C]acetyl chloride and tert-butylmagnesium chloride with cuprous chloride catalyst, followed by bromination. It was examined as an active-site directed label for acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AcChE). AcChE, isolated from Torpedo nobiliana, has kcat=(4.00±0.04)·103s−1, Km=0.055±0.008 mM in hydrolysis of acetylthiocholine, and kcat=(5.6±0.2)·103s−1, Km=0.051±0.003 mM in hydrolysis of acetylcholine. BrPin, binding in the trimethyl cavity, acts initially as a reversible competitive inhibitor, Ki=0.20±0.09 mM, and, with time, as an irreversible covalently bound inactivator. Introduction of 14C from [14C]BrPin into Torpedo AcChE at pH 7.0 was followed by SDS-PAGE, autoradiography and scintillation counting, in the absence and presence of 5-trimethylammonio-2-pentanone (TAP), a competitive inhibitor (Ki=0.075±0.001 mM) isosteric with acetylcholine; 1.8–1.914C was incorporated per inactivated enzyme unit at 50% inactivation. TAP retarded i of 0.9–1.114C per unit of enzyme protected. Prior inactivation of AcChE by BrPin prevents reaction with [3H]diisopropyl fluorophosphate ([3H]DFP). Prior inactivation by DFP or [3H]DFP does not prevent reaction with [14C]BrPin, and this subsequent reaction with BrPin does not displace the [3H] moiety. [14C]BrPin alkylates a nucleophile in the active site, and this reaction does not alkylate or utilize the serine-hydroxyl.

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