Resistance of butyrylcholinesterase to inactivation by ultrasound: Effects of ultrasound on catalytic activity and subunit association

Marie Thérèse Froment, Oksana Lockridge, Patrick Masson

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Abstract

The effects of 20 kHz ultrasound on catalytic activity and structure of the tetramer of wild-type human butyrylcholinesterase (BChE) from plasma and recombinant D70G mutant enzyme were studied at constant temperature. Effects on catalytic properties of both enzymes were investigated by kinetic analysis under ultrasound irradiation using a neutral substrate (o-nitrophenylbutyrate), a positively charged substrate (butyrylthiocholine), and a negatively charged substrate (aspirin). Effects on structure of highly purified wild-type BChE were followed by gel electrophoresis and activity measurements at V(max) after ultrasound treatment. Unlike hydrostatic pressure, mild ultrasound had moderate effects on catalytic parameters of BChE-catalyzed hydrolysis of substrates. For both wild-type and D70G, K(m) increased slightly with butyrylthiocholine and o-nitrophenylbutyrate under ultrasound irradiation, suggesting that these effects of ultrasound were not due to the periodic variation of pressure but rather to shear forces that took off substrate from the peripheral site and altered diffusion to the active site. By contrast, affinity of the D70G mutant for aspirin slightly increased with ultrasound power, suggesting that ultrasound-induced microstreaming unmasked peripheral residues involved in recognition and initial binding of the negatively charged substrate. Results support the contention that K(m) is a composite affinity constant, including dissociation constant of the first encounter enzyme-substrate complex on the peripheral site. Small changes in catalytic activity may have resulted from ultrasound-induced subtle conformational changes altering the active site reactivity. Short ultrasound irradiation induced a faint transient enzyme activation, but prolonged irradiation caused partial dissociation of the tetrameric enzyme and irreversible inactivation. Partial dissociation was related to enzyme microheterogeneity, i.e., nicked (C-terminal segment depleted) tetramers were less stable than native tetramers. The resistance of the native tetramer to ultrasound-induced dissociation was ascribed to the existence of an aromatic amino acid array on the apolar side of the C-terminal helical segment of subunits, the four subunits being held together in a four-helix bundle containing the aromatic zipper motifs. Aromatic/aromatic interactions between the four helical segments are thought to be enhanced by ultrasound-generated pressure. Copyright (C) 1998 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)53-64
Number of pages12
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1387
Issue number1-2
DOIs
StatePublished - Sep 8 1998

Fingerprint

Butyrylcholinesterase
Catalyst activity
Catalytic Domain
Ultrasonics
Association reactions
Butyrylthiocholine
Enzymes
Aspirin
Substrates
Pressure
Aromatic Amino Acids
Hydrostatic Pressure
Enzyme Activation
Irradiation
Electrophoresis
Hydrolysis
Gels
Temperature
Fasteners
Takeoff

Keywords

  • Aromatic zipper
  • Butyrylcholinesterase
  • Dissociation
  • Inactivation
  • Kinetics
  • Peripheral anionic site
  • Quaternary structure
  • Ultrasound

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

Cite this

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title = "Resistance of butyrylcholinesterase to inactivation by ultrasound: Effects of ultrasound on catalytic activity and subunit association",
abstract = "The effects of 20 kHz ultrasound on catalytic activity and structure of the tetramer of wild-type human butyrylcholinesterase (BChE) from plasma and recombinant D70G mutant enzyme were studied at constant temperature. Effects on catalytic properties of both enzymes were investigated by kinetic analysis under ultrasound irradiation using a neutral substrate (o-nitrophenylbutyrate), a positively charged substrate (butyrylthiocholine), and a negatively charged substrate (aspirin). Effects on structure of highly purified wild-type BChE were followed by gel electrophoresis and activity measurements at V(max) after ultrasound treatment. Unlike hydrostatic pressure, mild ultrasound had moderate effects on catalytic parameters of BChE-catalyzed hydrolysis of substrates. For both wild-type and D70G, K(m) increased slightly with butyrylthiocholine and o-nitrophenylbutyrate under ultrasound irradiation, suggesting that these effects of ultrasound were not due to the periodic variation of pressure but rather to shear forces that took off substrate from the peripheral site and altered diffusion to the active site. By contrast, affinity of the D70G mutant for aspirin slightly increased with ultrasound power, suggesting that ultrasound-induced microstreaming unmasked peripheral residues involved in recognition and initial binding of the negatively charged substrate. Results support the contention that K(m) is a composite affinity constant, including dissociation constant of the first encounter enzyme-substrate complex on the peripheral site. Small changes in catalytic activity may have resulted from ultrasound-induced subtle conformational changes altering the active site reactivity. Short ultrasound irradiation induced a faint transient enzyme activation, but prolonged irradiation caused partial dissociation of the tetrameric enzyme and irreversible inactivation. Partial dissociation was related to enzyme microheterogeneity, i.e., nicked (C-terminal segment depleted) tetramers were less stable than native tetramers. The resistance of the native tetramer to ultrasound-induced dissociation was ascribed to the existence of an aromatic amino acid array on the apolar side of the C-terminal helical segment of subunits, the four subunits being held together in a four-helix bundle containing the aromatic zipper motifs. Aromatic/aromatic interactions between the four helical segments are thought to be enhanced by ultrasound-generated pressure. Copyright (C) 1998 Elsevier Science B.V.",
keywords = "Aromatic zipper, Butyrylcholinesterase, Dissociation, Inactivation, Kinetics, Peripheral anionic site, Quaternary structure, Ultrasound",
author = "Froment, {Marie Th{\'e}r{\`e}se} and Oksana Lockridge and Patrick Masson",
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TY - JOUR

T1 - Resistance of butyrylcholinesterase to inactivation by ultrasound

T2 - Effects of ultrasound on catalytic activity and subunit association

AU - Froment, Marie Thérèse

AU - Lockridge, Oksana

AU - Masson, Patrick

PY - 1998/9/8

Y1 - 1998/9/8

N2 - The effects of 20 kHz ultrasound on catalytic activity and structure of the tetramer of wild-type human butyrylcholinesterase (BChE) from plasma and recombinant D70G mutant enzyme were studied at constant temperature. Effects on catalytic properties of both enzymes were investigated by kinetic analysis under ultrasound irradiation using a neutral substrate (o-nitrophenylbutyrate), a positively charged substrate (butyrylthiocholine), and a negatively charged substrate (aspirin). Effects on structure of highly purified wild-type BChE were followed by gel electrophoresis and activity measurements at V(max) after ultrasound treatment. Unlike hydrostatic pressure, mild ultrasound had moderate effects on catalytic parameters of BChE-catalyzed hydrolysis of substrates. For both wild-type and D70G, K(m) increased slightly with butyrylthiocholine and o-nitrophenylbutyrate under ultrasound irradiation, suggesting that these effects of ultrasound were not due to the periodic variation of pressure but rather to shear forces that took off substrate from the peripheral site and altered diffusion to the active site. By contrast, affinity of the D70G mutant for aspirin slightly increased with ultrasound power, suggesting that ultrasound-induced microstreaming unmasked peripheral residues involved in recognition and initial binding of the negatively charged substrate. Results support the contention that K(m) is a composite affinity constant, including dissociation constant of the first encounter enzyme-substrate complex on the peripheral site. Small changes in catalytic activity may have resulted from ultrasound-induced subtle conformational changes altering the active site reactivity. Short ultrasound irradiation induced a faint transient enzyme activation, but prolonged irradiation caused partial dissociation of the tetrameric enzyme and irreversible inactivation. Partial dissociation was related to enzyme microheterogeneity, i.e., nicked (C-terminal segment depleted) tetramers were less stable than native tetramers. The resistance of the native tetramer to ultrasound-induced dissociation was ascribed to the existence of an aromatic amino acid array on the apolar side of the C-terminal helical segment of subunits, the four subunits being held together in a four-helix bundle containing the aromatic zipper motifs. Aromatic/aromatic interactions between the four helical segments are thought to be enhanced by ultrasound-generated pressure. Copyright (C) 1998 Elsevier Science B.V.

AB - The effects of 20 kHz ultrasound on catalytic activity and structure of the tetramer of wild-type human butyrylcholinesterase (BChE) from plasma and recombinant D70G mutant enzyme were studied at constant temperature. Effects on catalytic properties of both enzymes were investigated by kinetic analysis under ultrasound irradiation using a neutral substrate (o-nitrophenylbutyrate), a positively charged substrate (butyrylthiocholine), and a negatively charged substrate (aspirin). Effects on structure of highly purified wild-type BChE were followed by gel electrophoresis and activity measurements at V(max) after ultrasound treatment. Unlike hydrostatic pressure, mild ultrasound had moderate effects on catalytic parameters of BChE-catalyzed hydrolysis of substrates. For both wild-type and D70G, K(m) increased slightly with butyrylthiocholine and o-nitrophenylbutyrate under ultrasound irradiation, suggesting that these effects of ultrasound were not due to the periodic variation of pressure but rather to shear forces that took off substrate from the peripheral site and altered diffusion to the active site. By contrast, affinity of the D70G mutant for aspirin slightly increased with ultrasound power, suggesting that ultrasound-induced microstreaming unmasked peripheral residues involved in recognition and initial binding of the negatively charged substrate. Results support the contention that K(m) is a composite affinity constant, including dissociation constant of the first encounter enzyme-substrate complex on the peripheral site. Small changes in catalytic activity may have resulted from ultrasound-induced subtle conformational changes altering the active site reactivity. Short ultrasound irradiation induced a faint transient enzyme activation, but prolonged irradiation caused partial dissociation of the tetrameric enzyme and irreversible inactivation. Partial dissociation was related to enzyme microheterogeneity, i.e., nicked (C-terminal segment depleted) tetramers were less stable than native tetramers. The resistance of the native tetramer to ultrasound-induced dissociation was ascribed to the existence of an aromatic amino acid array on the apolar side of the C-terminal helical segment of subunits, the four subunits being held together in a four-helix bundle containing the aromatic zipper motifs. Aromatic/aromatic interactions between the four helical segments are thought to be enhanced by ultrasound-generated pressure. Copyright (C) 1998 Elsevier Science B.V.

KW - Aromatic zipper

KW - Butyrylcholinesterase

KW - Dissociation

KW - Inactivation

KW - Kinetics

KW - Peripheral anionic site

KW - Quaternary structure

KW - Ultrasound

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U2 - 10.1016/S0167-4838(98)00105-8

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M3 - Article

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VL - 1387

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JO - Biochimica et Biophysica Acta - Proteins and Proteomics

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