Catalysis by acetylcholinesterase: evidence that the rate-limiting step for acylation with certain substrates precedes general acid-base catalysis

Proc Natl Acad Sci U S A. 1975 Oct;72(10):3834-8. doi: 10.1073/pnas.72.10.3834.


Inferences about the catalytic mechanism of acetylcholinesterase (acetylcholine hydrolase, EC are frequently made on the basis of a presumed analogy with chymotrypsin, EC Although both enzymes are serine hydrolases, several differences in the steady-state kinetic properties of the two have been observed. In this report particular attention is focused on the second-order reaction constant, kcat/Kapp. While the reported pH dependence and deuterium oxide isotope effect associated with this parameter for chymotrypsin are generally consistent with simple models involving rate-limiting general acid-base catalysis, this study finds a more complicated situation with acetylcholinesterase. The apparent pKa of kcat/Kapp for acetylcholinesterase varies between 5.5 and 6.3 for neutral substrates and involves nonlinear inhibition by [H+]. Deuterium oxide isotope effects for kcat/Kapp range from 1.1 for acetylcholine to 1.9 for p-nitrophenyl acetate. The bimolecular reaction rate appears rate-limiting for acetylcholine at low concentrations, while a rate-limiting induced-fit step is proposed to account for apparent pKa values and low deuterium oxide isotope effects associated with low concentrations of phenyl acetate and isoamyl acetate.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylcholine
  • Acetylcholinesterase / metabolism*
  • Animals
  • Binding Sites
  • Electric Organ / enzymology
  • Electrophorus
  • Hydrogen-Ion Concentration
  • Kinetics
  • Mathematics
  • Phenylacetates
  • Protein Binding
  • Structure-Activity Relationship


  • Phenylacetates
  • Acetylcholinesterase
  • Acetylcholine