Allosteric Control of Acetylcholinesterase Catalysis by Fasciculin (∗)

The interaction of fasciculin 2 was examined with wild-type and several mutant forms of acetylcholinesterase (AChE) where Trp86, which lies at the base of the active center gorge, is replaced by Tyr, Phe, and Ala. The fasciculin family of peptides from snake venom bind to a peripheral site near the...

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Bibliographic Details
Published in:The Journal of biological chemistry 1995-09, Vol.270 (35), p.20391-20399
Main Authors: Radić, Zoran, Quinn, Daniel M., Vellom, Daniel C., Camp, Shelley, Taylor, Palmer
Format: Article
Language:English
Subjects:
Acetylcholinesterase - chemistry
Acetylcholinesterase - metabolism
Alanine
Allosteric Regulation
Allosteric Site
Amino Acid Sequence
Animals
Catalysis
Cell Line
Cholinesterase Inhibitors - pharmacology
Elapid Venoms - metabolism
Elapid Venoms - pharmacology
Humans
Kinetics
Mathematics
Mice
Models, Theoretical
Mutagenesis, Site-Directed
Phenylalanine
Point Mutation
Protein Conformation
Protein Structure, Secondary
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Transfection
Tryptophan
Tyrosine
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Summary:The interaction of fasciculin 2 was examined with wild-type and several mutant forms of acetylcholinesterase (AChE) where Trp86, which lies at the base of the active center gorge, is replaced by Tyr, Phe, and Ala. The fasciculin family of peptides from snake venom bind to a peripheral site near the rim of the gorge, but at a position which still allows substrates and other inhibitors to enter the gorge. The interaction of a series of charged and uncharged carboxyl esters, alkyl phosphoryl esters, and substituted trifluoroacetophenones were analyzed with the wild-type and mutant AChEs in the presence and absence of fasciculin. We show that Trp86 is important for the alignment of carboxyl ester substrates in the AChE active center. The most marked influence of Trp86 substitution in inhibiting catalysis is seen for carboxyl esters that show rapid turnover. The extent of inhibition achieved with bound fasciculin is also greatest for efficiently catalyzed, charged substrates. When Ala is substituted for Trp86, fasciculin becomes an allosteric activator instead of an inhibitor for certain substrates. Analysis of the kinetics of acylation by organophosphates and conjugation by trifluoroacetophenones, along with deconstruction of the kinetic constants for carboxyl esters, suggests that AChE inhibition by fasciculin arises from reductions of both the commitment to catalysis and diffusional entry of substrate into the gorge. The former is reflected in the ratio of the rate constant for substrate acylation to that for dissociation of the initial complex. The action of fasciculin appears to be mediated allosterically from its binding site at the rim of the gorge to affect the orientation of the side chain of Trp86 which lies at the gorge base.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.270.35.20391