Substrate activation in acetylcholinesterase induced by low pH or mutation in the π-cation subsite

Patrick Masson, Lawrence M. Schopfer, Cynthia F. Bartels, Marie Thérèse Froment, Fabien Ribes, Florian Nachon, Oksana Lockridge

Research output: Contribution to journalArticlepeer-review

44 Scopus citations


Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the π-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the π-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.

Original languageEnglish (US)
Pages (from-to)313-324
Number of pages12
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Issue number2
StatePublished - Feb 11 2002


  • Acetylcholinesterase
  • Activation/inhibition by excess substrate
  • Butyrylcholinesterase
  • Catalytic triad
  • Conformational change
  • PH dependence
  • Peripheral anionic site

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology


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