TY - JOUR
T1 - Substrate activation in acetylcholinesterase induced by low pH or mutation in the π-cation subsite
AU - Masson, Patrick
AU - Schopfer, Lawrence M.
AU - Bartels, Cynthia F.
AU - Froment, Marie Thérèse
AU - Ribes, Fabien
AU - Nachon, Florian
AU - Lockridge, Oksana
N1 - Funding Information:
We thank Dr. C. Bon (Institut Pasteur, Paris, France) for the gift of the cDNA for B. fasciatus AChE and Dr. A. Shafferman (IIBR, Ness-Ziona, Israel) for the gift of the cDNA for human AChE. This work was supported by grant U.S. Army Medical Research and Materiel Command DAMD 17-97-1-7349 to O.L., grant DGA/OCDA 00-2-032-0-00 to O.L., and grant DSP/STTC(99CO 029/PEA) to P.M.
PY - 2002/2/11
Y1 - 2002/2/11
N2 - 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.
AB - 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.
KW - Acetylcholinesterase
KW - Activation/inhibition by excess substrate
KW - Butyrylcholinesterase
KW - Catalytic triad
KW - Conformational change
KW - PH dependence
KW - Peripheral anionic site
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U2 - 10.1016/S0167-4838(01)00323-5
DO - 10.1016/S0167-4838(01)00323-5
M3 - Article
C2 - 11904227
AN - SCOPUS:0037060466
SN - 0167-4838
VL - 1594
SP - 313
EP - 324
JO - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
JF - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
IS - 2
ER -