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.
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.
Salih
E
E
Department of Chemistry, Brandeis University, Waltham, MA 02254.
Chishti
S B
SB
Vicedomine
P
P
Cohen
S G
SG
Chiara
D C
DC
Cohen
J B
JB
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
D000345:Affinity Labels; D002074:Butanones; D002250:Carbon Radioisotopes; D010455:Peptides; D014316:Tritium; D007531:Isoflurophate; D006639:Histidine; C036537:1-bromopinacolone; D000110:Acetylcholinesterase; D014357:Trypsin