Tryptophan residue(s) as major components of the human serum paraoxonase active site

Denis Josse, Weihua Xie, Patrick Masson, Lawrence M Schopfer, Oksana Lockridge

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Serum paraoxonase (PON1, EC 3.1.8.1.) is a high density lipid- (HDL)-associated, calcium-dependent enzyme whose 3D structure, active site residues and physiological substrates are not known. The kinetic parameters k(cat) and K(m) (relative to k(cat) and K(m) of the wild-type), determined with four substrates (phenylacetate, paraoxon, diazoxon and chlorpyrifosoxon) were less than 1% and more than 100% for the W280A and W280F mutant enzymes, respectively. These results indicated that the aromatic/hydrophobic character of the amino acid in position 280 is essential for PON1 activity. In this study, we investigated whether this aromatic residue is in the PON1 active site. Group-specific labelling studies with N-bromosuccinimide, an oxidative agent of tryptophan, strongly suggested that one or several Trp could be in the active site of PON1 but we could not conclude either on the specificity of the labelling reaction or on the number of oxidized Trp. However, although PON activity was not altered by the hydrophilic tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl chloride (NBC), it was significantly reduced by the p-nitrophenylacetate analog 2-acetoxy-5-nitrobenzyl chloride (ANBC), whose hydrolysis by PON1 generated NBC in the active site. Moreover, since at least one calcium ion is present in the PON catalytic site, we attempted to probe the metal local environment using the calcium analog terbium. The luminescence spectrum of the PON-terbium complex exhibited an emission peak at 545 nm characteristic of an aromatic residue (Trp and/or Tyr)-terbium interaction. In conclusion, both the results obtained with the mechanism-based inhibitor of PON1 (ANBC) and the calcium-binding site luminescent probe terbium support the hypothesis of the presence of at least one Trp residue in the PON1 active site. Trp residue(s) may be involved in the binding of aromatic substrates. Copyright (C) 1999 Elsevier Science Ireland Ltd.

Original languageEnglish (US)
Pages (from-to)79-84
Number of pages6
JournalChemico-Biological Interactions
Volume119-120
DOIs
StatePublished - May 14 1999

Fingerprint

Terbium
Aryldialkylphosphatase
Tryptophan
Catalytic Domain
Calcium
Serum
Labeling
Chlorides
Substrates
Bromosuccinimide
Paraoxon
Calcium Chloride
Enzymes
Kinetic parameters
Luminescence
Hydrolysis
Metals
Binding Sites
Ions
Lipids

Keywords

  • Active site
  • Human serum paroxanase
  • Tryptophan residue(s)

ASJC Scopus subject areas

  • Toxicology

Cite this

Tryptophan residue(s) as major components of the human serum paraoxonase active site. / Josse, Denis; Xie, Weihua; Masson, Patrick; Schopfer, Lawrence M; Lockridge, Oksana.

In: Chemico-Biological Interactions, Vol. 119-120, 14.05.1999, p. 79-84.

Research output: Contribution to journalArticle

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abstract = "Serum paraoxonase (PON1, EC 3.1.8.1.) is a high density lipid- (HDL)-associated, calcium-dependent enzyme whose 3D structure, active site residues and physiological substrates are not known. The kinetic parameters k(cat) and K(m) (relative to k(cat) and K(m) of the wild-type), determined with four substrates (phenylacetate, paraoxon, diazoxon and chlorpyrifosoxon) were less than 1{\%} and more than 100{\%} for the W280A and W280F mutant enzymes, respectively. These results indicated that the aromatic/hydrophobic character of the amino acid in position 280 is essential for PON1 activity. In this study, we investigated whether this aromatic residue is in the PON1 active site. Group-specific labelling studies with N-bromosuccinimide, an oxidative agent of tryptophan, strongly suggested that one or several Trp could be in the active site of PON1 but we could not conclude either on the specificity of the labelling reaction or on the number of oxidized Trp. However, although PON activity was not altered by the hydrophilic tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl chloride (NBC), it was significantly reduced by the p-nitrophenylacetate analog 2-acetoxy-5-nitrobenzyl chloride (ANBC), whose hydrolysis by PON1 generated NBC in the active site. Moreover, since at least one calcium ion is present in the PON catalytic site, we attempted to probe the metal local environment using the calcium analog terbium. The luminescence spectrum of the PON-terbium complex exhibited an emission peak at 545 nm characteristic of an aromatic residue (Trp and/or Tyr)-terbium interaction. In conclusion, both the results obtained with the mechanism-based inhibitor of PON1 (ANBC) and the calcium-binding site luminescent probe terbium support the hypothesis of the presence of at least one Trp residue in the PON1 active site. Trp residue(s) may be involved in the binding of aromatic substrates. Copyright (C) 1999 Elsevier Science Ireland Ltd.",
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T1 - Tryptophan residue(s) as major components of the human serum paraoxonase active site

AU - Josse, Denis

AU - Xie, Weihua

AU - Masson, Patrick

AU - Schopfer, Lawrence M

AU - Lockridge, Oksana

PY - 1999/5/14

Y1 - 1999/5/14

N2 - Serum paraoxonase (PON1, EC 3.1.8.1.) is a high density lipid- (HDL)-associated, calcium-dependent enzyme whose 3D structure, active site residues and physiological substrates are not known. The kinetic parameters k(cat) and K(m) (relative to k(cat) and K(m) of the wild-type), determined with four substrates (phenylacetate, paraoxon, diazoxon and chlorpyrifosoxon) were less than 1% and more than 100% for the W280A and W280F mutant enzymes, respectively. These results indicated that the aromatic/hydrophobic character of the amino acid in position 280 is essential for PON1 activity. In this study, we investigated whether this aromatic residue is in the PON1 active site. Group-specific labelling studies with N-bromosuccinimide, an oxidative agent of tryptophan, strongly suggested that one or several Trp could be in the active site of PON1 but we could not conclude either on the specificity of the labelling reaction or on the number of oxidized Trp. However, although PON activity was not altered by the hydrophilic tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl chloride (NBC), it was significantly reduced by the p-nitrophenylacetate analog 2-acetoxy-5-nitrobenzyl chloride (ANBC), whose hydrolysis by PON1 generated NBC in the active site. Moreover, since at least one calcium ion is present in the PON catalytic site, we attempted to probe the metal local environment using the calcium analog terbium. The luminescence spectrum of the PON-terbium complex exhibited an emission peak at 545 nm characteristic of an aromatic residue (Trp and/or Tyr)-terbium interaction. In conclusion, both the results obtained with the mechanism-based inhibitor of PON1 (ANBC) and the calcium-binding site luminescent probe terbium support the hypothesis of the presence of at least one Trp residue in the PON1 active site. Trp residue(s) may be involved in the binding of aromatic substrates. Copyright (C) 1999 Elsevier Science Ireland Ltd.

AB - Serum paraoxonase (PON1, EC 3.1.8.1.) is a high density lipid- (HDL)-associated, calcium-dependent enzyme whose 3D structure, active site residues and physiological substrates are not known. The kinetic parameters k(cat) and K(m) (relative to k(cat) and K(m) of the wild-type), determined with four substrates (phenylacetate, paraoxon, diazoxon and chlorpyrifosoxon) were less than 1% and more than 100% for the W280A and W280F mutant enzymes, respectively. These results indicated that the aromatic/hydrophobic character of the amino acid in position 280 is essential for PON1 activity. In this study, we investigated whether this aromatic residue is in the PON1 active site. Group-specific labelling studies with N-bromosuccinimide, an oxidative agent of tryptophan, strongly suggested that one or several Trp could be in the active site of PON1 but we could not conclude either on the specificity of the labelling reaction or on the number of oxidized Trp. However, although PON activity was not altered by the hydrophilic tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl chloride (NBC), it was significantly reduced by the p-nitrophenylacetate analog 2-acetoxy-5-nitrobenzyl chloride (ANBC), whose hydrolysis by PON1 generated NBC in the active site. Moreover, since at least one calcium ion is present in the PON catalytic site, we attempted to probe the metal local environment using the calcium analog terbium. The luminescence spectrum of the PON-terbium complex exhibited an emission peak at 545 nm characteristic of an aromatic residue (Trp and/or Tyr)-terbium interaction. In conclusion, both the results obtained with the mechanism-based inhibitor of PON1 (ANBC) and the calcium-binding site luminescent probe terbium support the hypothesis of the presence of at least one Trp residue in the PON1 active site. Trp residue(s) may be involved in the binding of aromatic substrates. Copyright (C) 1999 Elsevier Science Ireland Ltd.

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