title: Active-site peptides of acetylcholinesterase of Electrophorus electricus: labelling of His-440 by 1-bromo-[2-14C]pinacolone and Ser-200 by tritiated diisopropyl fluorophosphate.
abstract: To characterize the structure of the active site of acetylcholinesterase (AChE) from the electric organ of E. electricus, we identified sites of incorporation of two active-site affinity labels, [3H]diisopropyl fluorophosphate ([3H]DFP), and 1-bromo-2-[14C]pinacolone ([14C]BrPin). AChE was isolated, purified, inactivated and digested with trypsin, and peptides containing 3H or 14C were purified by reverse-phase HPLC and characterized by N-terminal sequence analysis. [3H]DFP, labelling Ser-200, was found in a single peptide, QVTIFGESAGAASVGMHLLSPDSR, 83% identical with the sequence from Thr-193 to Arg-216 deduced for AChE of T. californica, with Gln, Ala, Leu, and Asp in place of Thr-193, Gly-203, Ile-210 and Gly-214, respectively, and 87% identical with that from bovine and human brain AChEs. Inactivation by [14C]BrPin led to two radioactive peptides. One, ASNLVWPEWMGVIHGYEIEFVFGLPLEK, was 96% identical with that extending from Ala-427 to Lys-454 of T. californica. Release of 14C in cycle 14 established reaction of [14C]BrPin with active-site His-440, protected by 5-trimethylammonio-2-pentanone (TAP). The other peptide, LLXVTENIDDAER, 77% homologous with that of T. californica extending from Leu-531 to Arg-543, had label associated with the third cycle, not protected by TAP, corresponding to Asn-533. The slow inactivation of eel AChE by reaction of [14C]BrPin at His-440 contrasts with that of AChE from T. nobiliana, where it reacts rapidly with a free cysteine, Cys-231, not present in eel AChE. For both AChEs, inactivation by BrPin prevents subsequent reaction with [3H]DFP, and prior inactivation by DFP does not prevent reactions with [14C]BrPin.
authors.affiliation: Department of Chemistry, Brandeis University, Waltham, MA 02254.
authors.forename: E
authors.initials: E
authors.lastname: Salih
authors.forename: S B
authors.initials: SB
authors.lastname: Chishti
authors.forename: P
authors.initials: P
authors.lastname: Vicedomine
authors.forename: S G
authors.initials: SG
authors.lastname: Cohen
authors.forename: D C
authors.initials: DC
authors.lastname: Chiara
authors.forename: J B
authors.initials: JB
authors.lastname: Cohen
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry / Q000302:isolation & purification*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002074:Butanones; D002250:Carbon Radioisotopes; D004593:Electrophorus / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D007531:Isoflurophate; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D014316:Tritium; D014357:Trypsin
chemical_list: D000345:Affinity Labels; D002074:Butanones; D002250:Carbon Radioisotopes; D010455:Peptides; D014316:Tritium; D007531:Isoflurophate; D006639:Histidine; C036537:1-bromopinacolone; D000110:Acetylcholinesterase; D014357:Trypsin