Asp70 in the peripheral anionic site of human butyrylcholinesterase

Patrick Masson, Marie Thérèse Froment, Cynthia F. Bartels, Oksana Lockridge

Research output: Contribution to journalArticle

109 Citations (Scopus)

Abstract

The goal of this work was to determine what amino acids at the mouth of the active-site gorge are important for the function of human butyrylcholinesterase. Mutants D70G, Q119Y, G283D, A277W, A277H and A277W/G283D were expressed in human embryonal kidney cells and the secreted enzymes were assayed by steady-state kinetics. The result was that only one amino acid, D70 was found to be important for function. When D70 was mutated to G, the same mutation as in the naturally occurring atypical butyrylcholinesterase, the affinity for positively charged substrates and positively charged inhibitors decreased 5-30-fold. The D70G mutant had another striking abnormality in that it was virtually devoid of the phenomenon of substrate activation by excess butyrylthiocholine. Thus, though k(cat) was the same for wild-type and D70G mutant, being 24000 min-1 at low butyrylthiocholine concentrations (0.01-0.1 mM), it failed to increase for the D70G mutant at 40 mM butyrylthiocholine, whereas it increased three-fold for wild type. The D70G mutant was more sensitive to changes in salt concentration, its catalytic rate decreasing more than that of the wild type. The D70G mutant appeared to have a greater surface negative charge than wild type suggesting that the D70G mutant had a conformation different from that of the wild type. That D70 affects the function of butyrylcholinesterase, together with its location at the mouth of the active-site gorge, supports the hypothesis that D70 is a component of the peripheral anionic site of butyrylcholinesterase. Mutants containing aromatic amino acids at the mouth of the gorge had increased binding affinity for propidium and fasciculin, but unaltered function, suggesting that aromatic amino acids are not important to the function of the peripheral anionic site of butyrylcholinesterase.

Original languageEnglish (US)
Pages (from-to)36-48
Number of pages13
JournalEuropean Journal of Biochemistry
Volume235
Issue number1-2
DOIs
StatePublished - Jan 1 1996

Fingerprint

Butyrylcholinesterase
Butyrylthiocholine
Mouth
Aromatic Amino Acids
Catalytic Domain
Amino Acids
Propidium
Substrates
Conformations
Salts
Chemical activation
Kidney
Mutation
Kinetics
Enzymes

Keywords

  • aspartate 70
  • atypical
  • butyrylcholinesterase
  • peripheral anionic site
  • propidium

ASJC Scopus subject areas

  • Biochemistry

Cite this

Asp70 in the peripheral anionic site of human butyrylcholinesterase. / Masson, Patrick; Froment, Marie Thérèse; Bartels, Cynthia F.; Lockridge, Oksana.

In: European Journal of Biochemistry, Vol. 235, No. 1-2, 01.01.1996, p. 36-48.

Research output: Contribution to journalArticle

Masson, Patrick ; Froment, Marie Thérèse ; Bartels, Cynthia F. ; Lockridge, Oksana. / Asp70 in the peripheral anionic site of human butyrylcholinesterase. In: European Journal of Biochemistry. 1996 ; Vol. 235, No. 1-2. pp. 36-48.
@article{8d306f0d17a5458cb517969476815310,
title = "Asp70 in the peripheral anionic site of human butyrylcholinesterase",
abstract = "The goal of this work was to determine what amino acids at the mouth of the active-site gorge are important for the function of human butyrylcholinesterase. Mutants D70G, Q119Y, G283D, A277W, A277H and A277W/G283D were expressed in human embryonal kidney cells and the secreted enzymes were assayed by steady-state kinetics. The result was that only one amino acid, D70 was found to be important for function. When D70 was mutated to G, the same mutation as in the naturally occurring atypical butyrylcholinesterase, the affinity for positively charged substrates and positively charged inhibitors decreased 5-30-fold. The D70G mutant had another striking abnormality in that it was virtually devoid of the phenomenon of substrate activation by excess butyrylthiocholine. Thus, though k(cat) was the same for wild-type and D70G mutant, being 24000 min-1 at low butyrylthiocholine concentrations (0.01-0.1 mM), it failed to increase for the D70G mutant at 40 mM butyrylthiocholine, whereas it increased three-fold for wild type. The D70G mutant was more sensitive to changes in salt concentration, its catalytic rate decreasing more than that of the wild type. The D70G mutant appeared to have a greater surface negative charge than wild type suggesting that the D70G mutant had a conformation different from that of the wild type. That D70 affects the function of butyrylcholinesterase, together with its location at the mouth of the active-site gorge, supports the hypothesis that D70 is a component of the peripheral anionic site of butyrylcholinesterase. Mutants containing aromatic amino acids at the mouth of the gorge had increased binding affinity for propidium and fasciculin, but unaltered function, suggesting that aromatic amino acids are not important to the function of the peripheral anionic site of butyrylcholinesterase.",
keywords = "aspartate 70, atypical, butyrylcholinesterase, peripheral anionic site, propidium",
author = "Patrick Masson and Froment, {Marie Th{\'e}r{\`e}se} and Bartels, {Cynthia F.} and Oksana Lockridge",
year = "1996",
month = "1",
day = "1",
doi = "10.1111/j.1432-1033.1996.00036.x",
language = "English (US)",
volume = "235",
pages = "36--48",
journal = "FEBS Journal",
issn = "1742-464X",
publisher = "Wiley-Blackwell",
number = "1-2",

}

TY - JOUR

T1 - Asp70 in the peripheral anionic site of human butyrylcholinesterase

AU - Masson, Patrick

AU - Froment, Marie Thérèse

AU - Bartels, Cynthia F.

AU - Lockridge, Oksana

PY - 1996/1/1

Y1 - 1996/1/1

N2 - The goal of this work was to determine what amino acids at the mouth of the active-site gorge are important for the function of human butyrylcholinesterase. Mutants D70G, Q119Y, G283D, A277W, A277H and A277W/G283D were expressed in human embryonal kidney cells and the secreted enzymes were assayed by steady-state kinetics. The result was that only one amino acid, D70 was found to be important for function. When D70 was mutated to G, the same mutation as in the naturally occurring atypical butyrylcholinesterase, the affinity for positively charged substrates and positively charged inhibitors decreased 5-30-fold. The D70G mutant had another striking abnormality in that it was virtually devoid of the phenomenon of substrate activation by excess butyrylthiocholine. Thus, though k(cat) was the same for wild-type and D70G mutant, being 24000 min-1 at low butyrylthiocholine concentrations (0.01-0.1 mM), it failed to increase for the D70G mutant at 40 mM butyrylthiocholine, whereas it increased three-fold for wild type. The D70G mutant was more sensitive to changes in salt concentration, its catalytic rate decreasing more than that of the wild type. The D70G mutant appeared to have a greater surface negative charge than wild type suggesting that the D70G mutant had a conformation different from that of the wild type. That D70 affects the function of butyrylcholinesterase, together with its location at the mouth of the active-site gorge, supports the hypothesis that D70 is a component of the peripheral anionic site of butyrylcholinesterase. Mutants containing aromatic amino acids at the mouth of the gorge had increased binding affinity for propidium and fasciculin, but unaltered function, suggesting that aromatic amino acids are not important to the function of the peripheral anionic site of butyrylcholinesterase.

AB - The goal of this work was to determine what amino acids at the mouth of the active-site gorge are important for the function of human butyrylcholinesterase. Mutants D70G, Q119Y, G283D, A277W, A277H and A277W/G283D were expressed in human embryonal kidney cells and the secreted enzymes were assayed by steady-state kinetics. The result was that only one amino acid, D70 was found to be important for function. When D70 was mutated to G, the same mutation as in the naturally occurring atypical butyrylcholinesterase, the affinity for positively charged substrates and positively charged inhibitors decreased 5-30-fold. The D70G mutant had another striking abnormality in that it was virtually devoid of the phenomenon of substrate activation by excess butyrylthiocholine. Thus, though k(cat) was the same for wild-type and D70G mutant, being 24000 min-1 at low butyrylthiocholine concentrations (0.01-0.1 mM), it failed to increase for the D70G mutant at 40 mM butyrylthiocholine, whereas it increased three-fold for wild type. The D70G mutant was more sensitive to changes in salt concentration, its catalytic rate decreasing more than that of the wild type. The D70G mutant appeared to have a greater surface negative charge than wild type suggesting that the D70G mutant had a conformation different from that of the wild type. That D70 affects the function of butyrylcholinesterase, together with its location at the mouth of the active-site gorge, supports the hypothesis that D70 is a component of the peripheral anionic site of butyrylcholinesterase. Mutants containing aromatic amino acids at the mouth of the gorge had increased binding affinity for propidium and fasciculin, but unaltered function, suggesting that aromatic amino acids are not important to the function of the peripheral anionic site of butyrylcholinesterase.

KW - aspartate 70

KW - atypical

KW - butyrylcholinesterase

KW - peripheral anionic site

KW - propidium

UR - http://www.scopus.com/inward/record.url?scp=0029665127&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029665127&partnerID=8YFLogxK

U2 - 10.1111/j.1432-1033.1996.00036.x

DO - 10.1111/j.1432-1033.1996.00036.x

M3 - Article

C2 - 8631355

AN - SCOPUS:0029665127

VL - 235

SP - 36

EP - 48

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 1-2

ER -