Labeling of cysteine 231 in acetylcholinesterase from Torpedo nobiliana by the active-site directed reagent, 1-bromo-2-[14C] pinacolone. Effects of 2,2'-dipyridyl disulfide and other sulfhydryl reagents.
Acetylcholinesterase (AcChE, EC 3.1.1.7) was isolated from the electric organ of T. nobiliana and treated with the active-site-directed alkylating agent 1-bromo-2-[14C]pinacolone ([14C]BrPin), or with BrPin, which acts initially as a competitive inhibitor, Ki = 0.18 mM, and then inactivates the enzyme, k2 = 1.8 x 10(-4) s-1. AcChE aliquots were digested with trypsin and fractionated by reversed phase high performance liquid chromatography. Inactivation caused a decrease in one absorption peak and an increase in another, identified as the peptide beginning at Ala-222 and extending to Arg-242. 5-Trimethylammonio-2-pentanone, a competitive inhibitor, isosteric with acetylcholine, retarded the inactivation and decreased the quantity of labeled peptide. On sequencing, the 14C label was found associated with Cys-231. This was confirmed by comparison with synthesized S-pinacolonylcysteine, by study of effects of blocking the sequencing by o-phthalaldehyde, and by inactivation by 2,2'-dipyridyl disulfide (2-PDS), a thiol-specific reagent that acts initially as a competitive inhibitor, Ki = 0.042 mM, and then inactivates the enzyme, k2 = 5.0 x 10(-4) s-1. This is retarded by 5-trimethylammonio-2-pentanone, and prior inactivation by 2-PDS prevents subsequent reaction of [14C]BrPin in the active site. BrPin inactivates AcChEs from Electrophorus electricus and from human erythrocyte, but 2-PDS does not. Neither reagent inactivates butyrylcholinesterases from human and horse serum.
Salih
E
E
Department of Chemistry, Brandeis University, Waltham, Massachusetts 02254.
Howard
S
S
Chishti
S B
SB
Cohen
S G
SG
Liu
W S
WS
Cohen
J B
JB
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives* / Q000494:pharmacology; D000110:Acetylcholinesterase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002074:Butanones / Q000378:metabolism* / Q000494:pharmacology; D002250:Carbon Radioisotopes; D002800:Cholinesterase Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004220:Disulfides / Q000494:pharmacology*; D004557:Electric Organ / Q000201:enzymology*; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014101:Torpedo; D014357:Trypsin
D002074:Butanones; D002250:Carbon Radioisotopes; D002800:Cholinesterase Inhibitors; D004220:Disulfides; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; C001105:2,2'-dipyridyl disulfide; C036537:1-bromopinacolone; D015082:2,2'-Dipyridyl; D000110:Acetylcholinesterase; D014357:Trypsin; D003545:Cysteine