The Binding Sites of Inhibitory Monoclonal Antibodies on Acetylcholinesterase

Abstract

We investigated the target sites of three inhibitory monoclonal antibodies on Electrophorus acetylcholinesterase (AChE). Previous studies showed that Elec-403 and Elec-410 are directed to overlapping but distinct epitopes in the peripheral site, at the entrance of the catalytic gorge, whereas Elec-408 binds to a different region. Using Electrophorus/rat AChE chimeras, we identified surface residues that differed between sensitive and insensitive AChEs: the replacement of a single Electrophorus residue by its rat homolog was able to abolish binding and inhibition, for each antibody. Reciprocally, binding and inhibition by Elec-403 and by Elec-410 could be conferred to rat AChE by the reverse mutation. Elec-410 appears to bind to one side of the active gorge, whereas Elec-403 covers its opening, explaining why the AChE-Elec-410 complex reacts faster than the AChE-Elec-403 or AChE-fasciculin complexes with two active site inhibitors, m-(N,N,N-trimethyltammonio)trifluoro-acetophenone and echothiophate. Elec-408 binds to the region of the putative "back door," distant from the peripheral site, and does not interfere with the access of inhibitors to the active site. The binding of an antibody to this novel regulatory site may inhibit the enzyme by blocking the back door or by inducing a conformational distortion within the active site.

Substrate and Product Trafficking through the Active Center Gorge of Acetylcholinesterase Analyzed by Crystallography and Equilibrium Binding

“…This suggests that this region does not provide an exit for the reaction products. Finally, an inhibitory antibody raised against Electrophorus electricus AChE was shown to bind to a surface site distinct from the PAS, and further identified as the region of the putative back door (63). In fact, the mAChE Asp 460 -Gln 474 segment that contains several SCh-and ATCh-interacting residues (Fig.…”

“…In fact, the mAChE Asp 460 -Gln 474 segment that contains several SCh-and ATCh-interacting residues (Fig. 4), corresponds to the E. electricus AChE segment Glu 484 -Arg 498 shown, by mutagenesis analysis, to be involved in antibody binding (63). That binding of this antibody does not prevent inhibition by the substrate, as shown by the unaltered K ss value, appears consistent with the absence of bound ACh in the back door region of the S203A mutant or bound substrate in this region on mAChE.…”

Acetylcholinesterase: Mechanism of Catalysis and Inhibition

“…The binding of antibodies decreased the kcat for the substrate hydrolysis by AChE and the antibodies partially competed with propidium and fasciculin for the binding site [112,113]. The mechanism of inhibition by antibodies could be similar to that of other non-competitive inhibitors, i.e.…”

“…It has been shown that monoclonal antibodies raised against phosphorylated AChE affect the catalytic activity of the enzyme by binding to a conformational epitope located at or near the active site gorge entrance [112,113]. The binding of antibodies decreased the kcat for the substrate hydrolysis by AChE and the antibodies partially competed with propidium and fasciculin for the binding site [112,113].…”

Post-translational modifications of proteins: Acetylcholinesterase as a model system

“…It has been proposed that binding to the peripheral site of AChE molecule induces allosteric modification of the orientation of Trp-84, which serves as the choline binding site [95]. It was also demonstrated that some antibodies can act as noncompetitive inhibitors, presumably through an allosteric mechanism [96].…”