Reactions of 1-bromo-2-[14C]pinacolone with acetylcholinesterase from Torpedo nobiliana. Effects of 5-trimethylammonio-2-pentanone and diisopropyl fluorophosphate.
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 k(cat) = (4.00 +/- 0.04).10(3) s-1, Km = 0.055 +/- 0.008 mM in hydrolysis of acetylthiocholine, and k(cat) = (5.6 +/- 0.2).10(3) s-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; 1.8-1.9 14C was incorporated per inactivated enzyme unit at 50% inactivation. TAP retarded inactivation by [14C]BrPin, and prevented introduction of 0.9-1.1 14C 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.
Department of Chemistry, Brandeis University, Waltham, MA 02254.
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Cohen
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Salih
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Solomon
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Howard
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Chishti
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Cohen
D000109:Acetylcholine / Q000031:analogs & derivatives*; D000110:Acetylcholinesterase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002074:Butanones / Q000138:chemical synthesis / Q000378:metabolism*; D002250:Carbon Radioisotopes; D004557:Electric Organ / Q000201:enzymology*; D007531:Isoflurophate / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D014101:Torpedo
D002074:Butanones; D002250:Carbon Radioisotopes; D000644:Quaternary Ammonium Compounds; D007531:Isoflurophate; C034175:trimethyl(4-oxopentyl)ammonium; C036537:1-bromopinacolone; D000110:Acetylcholinesterase; D000109:Acetylcholine