ACTIVE-SITE PEPTIDES OF ACETYLCHOLINESTERASE OF ELECTROPHORUS-ELECTRICUS - LABELING OF HIS-440 BY 1-BROMO-[2-C-14]PINACOLONE AND SER-200 BYTRITIATED DIISOPROPYL FLUOROPHOSPHATE

To characterize the structure of the active site of acetylcholinestera se (AChE) from the electric organ of E. electricus, we identified site s of incorporation of two active-site affinity labels, [H-3]diisopropy l fluorophosphate ([H-3]DFP), and 1-bromo-2-[C-14]pinacolone ([C-14]Br Pin). AChE was isolated, purified, inactivated and digested with tryps in, and peptides containing H-3 or C-14 were purified by reverse-phase HPLC and characterized by N-terminal sequence analysis. [H-3]DFP, lab elling Ser-200, was found in a single peptide, QVTIFGESAGAASVGMHLLSPDS R, 83% identical with the sequence from Thr-193 to Arg-216 deduced for AChE of T. californica, with Gin, Ala, Leu, and Asp in place of Thr-1 93, Gly-203, Ile-210 and Gly-214, respectively, and 87% identical with that from bovine and human brain AChEs. Inactivation by [C-14]BrPin l ed to two radioactive peptides. One, ASNLVWPEWMGVIHGYEIEFVFGLPLEK, was 96% identical with that extending from Ala-427 to Lys-454 of T. calif ornica. Release of C-14 in cycle 14 established reaction of [C-14]BrPi n with active-site His-440, protected by 5-trimethylammonio-2-pentanon e (TAP). The other peptide, LLXVTENIDDAER, 77% homologous with that of T. californica extending from Leu-531 to Arg-543, had label associate d with the third cycle, not protected by TAP, corresponding to Asn-533 . The slow inactivation of eel AChE by reaction of [C-14]BrPin at His- 440 contrasts with that of AChE from T. nobiliana, where it reacts rap idly with a free cysteine, Cys-231, not present in eel AChE. For both AChEs, inactivation by BrPin prevents subsequent reaction with [H-3]DF P, and prior inactivation by DFP does not prevent reactions with [C-14 ]BrPin.