Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant

Vladislav Levitsky, Weihua Xie, Marie Thérèse Froment, Oksana Lockridge, Patrick Masson

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Wild-type human butyrylcholinesterase (BuChE) has a non-Michaelian behaviour showing substrate activation with butyrylthiocholine (BTC) as the substrate. The D70G mutant has a catalytic constant identical to that of the wild-type enzyme, but a 10-fold lower affinity for BTC compared to wild-type enzyme, and it does not exhibit activation by excess BTC under conventional conditions. In the present work it was found that addition of polyols or sugars changed the kinetic behaviour of the D70G mutant with BTC. In the presence of 40% sucrose, the D70G mutant enzyme displayed marked activation by excess substrate. Because D70 is hydrogen bonded to Y332, mutants of Y332 were studied. Mutant Y332F had a behaviour similar to that of wild-type BuChE, whereas mutants Y332A, Y332A/D70G and D70G had negligible substrate activation. The behaviour of wild-type, Y332F, Y332A and Y332A/D70G did not change in the presence of high concentrations of sugar. Substrate activation has been explained by binding of a second substrate molecule in the peripheral site at D70. The D70G mutant should be incapable of substrate activation, if D70 were the only residue involved in substrate activation. The ability of the D70G mutant to display substrate activation by medium engineering suggests that other residues are involved in initial substrate binding and activation by excess substrate. Osmolyte-induced change in conformation and/or hydration status of Y332 and other solvent-exposed residues may account for the non-Michaelian behaviour of the D70G mutant. Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)422-430
Number of pages9
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1429
Issue number2
DOIs
StatePublished - Jan 11 1999

Fingerprint

Butyrylthiocholine
Butyrylcholinesterase
Chemical activation
Substrates
Enzymes
Aptitude
Sucrose
Hydrogen
Sugars
polyol
Hydration

Keywords

  • Butyrylcholinesterase
  • Medium engineering
  • Mutant
  • Peripheral anionic site
  • Substrate activation

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

Cite this

Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant. / Levitsky, Vladislav; Xie, Weihua; Froment, Marie Thérèse; Lockridge, Oksana; Masson, Patrick.

In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, Vol. 1429, No. 2, 11.01.1999, p. 422-430.

Research output: Contribution to journalArticle

Levitsky, Vladislav ; Xie, Weihua ; Froment, Marie Thérèse ; Lockridge, Oksana ; Masson, Patrick. / Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant. In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology. 1999 ; Vol. 1429, No. 2. pp. 422-430.
@article{794224abaab44c8b96460a2e22cac849,
title = "Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant",
abstract = "Wild-type human butyrylcholinesterase (BuChE) has a non-Michaelian behaviour showing substrate activation with butyrylthiocholine (BTC) as the substrate. The D70G mutant has a catalytic constant identical to that of the wild-type enzyme, but a 10-fold lower affinity for BTC compared to wild-type enzyme, and it does not exhibit activation by excess BTC under conventional conditions. In the present work it was found that addition of polyols or sugars changed the kinetic behaviour of the D70G mutant with BTC. In the presence of 40{\%} sucrose, the D70G mutant enzyme displayed marked activation by excess substrate. Because D70 is hydrogen bonded to Y332, mutants of Y332 were studied. Mutant Y332F had a behaviour similar to that of wild-type BuChE, whereas mutants Y332A, Y332A/D70G and D70G had negligible substrate activation. The behaviour of wild-type, Y332F, Y332A and Y332A/D70G did not change in the presence of high concentrations of sugar. Substrate activation has been explained by binding of a second substrate molecule in the peripheral site at D70. The D70G mutant should be incapable of substrate activation, if D70 were the only residue involved in substrate activation. The ability of the D70G mutant to display substrate activation by medium engineering suggests that other residues are involved in initial substrate binding and activation by excess substrate. Osmolyte-induced change in conformation and/or hydration status of Y332 and other solvent-exposed residues may account for the non-Michaelian behaviour of the D70G mutant. Copyright (C) 1999 Elsevier Science B.V.",
keywords = "Butyrylcholinesterase, Medium engineering, Mutant, Peripheral anionic site, Substrate activation",
author = "Vladislav Levitsky and Weihua Xie and Froment, {Marie Th{\'e}r{\`e}se} and Oksana Lockridge and Patrick Masson",
year = "1999",
month = "1",
day = "11",
doi = "10.1016/S0167-4838(98)00253-2",
language = "English (US)",
volume = "1429",
pages = "422--430",
journal = "Biochimica et Biophysica Acta - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant

AU - Levitsky, Vladislav

AU - Xie, Weihua

AU - Froment, Marie Thérèse

AU - Lockridge, Oksana

AU - Masson, Patrick

PY - 1999/1/11

Y1 - 1999/1/11

N2 - Wild-type human butyrylcholinesterase (BuChE) has a non-Michaelian behaviour showing substrate activation with butyrylthiocholine (BTC) as the substrate. The D70G mutant has a catalytic constant identical to that of the wild-type enzyme, but a 10-fold lower affinity for BTC compared to wild-type enzyme, and it does not exhibit activation by excess BTC under conventional conditions. In the present work it was found that addition of polyols or sugars changed the kinetic behaviour of the D70G mutant with BTC. In the presence of 40% sucrose, the D70G mutant enzyme displayed marked activation by excess substrate. Because D70 is hydrogen bonded to Y332, mutants of Y332 were studied. Mutant Y332F had a behaviour similar to that of wild-type BuChE, whereas mutants Y332A, Y332A/D70G and D70G had negligible substrate activation. The behaviour of wild-type, Y332F, Y332A and Y332A/D70G did not change in the presence of high concentrations of sugar. Substrate activation has been explained by binding of a second substrate molecule in the peripheral site at D70. The D70G mutant should be incapable of substrate activation, if D70 were the only residue involved in substrate activation. The ability of the D70G mutant to display substrate activation by medium engineering suggests that other residues are involved in initial substrate binding and activation by excess substrate. Osmolyte-induced change in conformation and/or hydration status of Y332 and other solvent-exposed residues may account for the non-Michaelian behaviour of the D70G mutant. Copyright (C) 1999 Elsevier Science B.V.

AB - Wild-type human butyrylcholinesterase (BuChE) has a non-Michaelian behaviour showing substrate activation with butyrylthiocholine (BTC) as the substrate. The D70G mutant has a catalytic constant identical to that of the wild-type enzyme, but a 10-fold lower affinity for BTC compared to wild-type enzyme, and it does not exhibit activation by excess BTC under conventional conditions. In the present work it was found that addition of polyols or sugars changed the kinetic behaviour of the D70G mutant with BTC. In the presence of 40% sucrose, the D70G mutant enzyme displayed marked activation by excess substrate. Because D70 is hydrogen bonded to Y332, mutants of Y332 were studied. Mutant Y332F had a behaviour similar to that of wild-type BuChE, whereas mutants Y332A, Y332A/D70G and D70G had negligible substrate activation. The behaviour of wild-type, Y332F, Y332A and Y332A/D70G did not change in the presence of high concentrations of sugar. Substrate activation has been explained by binding of a second substrate molecule in the peripheral site at D70. The D70G mutant should be incapable of substrate activation, if D70 were the only residue involved in substrate activation. The ability of the D70G mutant to display substrate activation by medium engineering suggests that other residues are involved in initial substrate binding and activation by excess substrate. Osmolyte-induced change in conformation and/or hydration status of Y332 and other solvent-exposed residues may account for the non-Michaelian behaviour of the D70G mutant. Copyright (C) 1999 Elsevier Science B.V.

KW - Butyrylcholinesterase

KW - Medium engineering

KW - Mutant

KW - Peripheral anionic site

KW - Substrate activation

UR - http://www.scopus.com/inward/record.url?scp=0032946621&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032946621&partnerID=8YFLogxK

U2 - 10.1016/S0167-4838(98)00253-2

DO - 10.1016/S0167-4838(98)00253-2

M3 - Article

C2 - 9989227

AN - SCOPUS:0032946621

VL - 1429

SP - 422

EP - 430

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 2

ER -