Nitro-fatty acid reaction with glutathione and cysteine: Kinetic analysis of thiol alkylation by a Michael addition reaction

Laura M.S. Baker, Paul R.S. Baker, Franca Golin-Bisello, Francisco J. Schopfer, Mitchell Fink, Steven R. Woodcock, Bruce Branchaud, Rafael Radi, Bruce A. Freeman

Research output: Contribution to journalArticle

128 Citations (Scopus)

Abstract

Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO2) and linoleic acid (LNO2) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO2 and LNO2 reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M-1s-1 and 355 M-1s -1, respectively, at pH 7.4 and 37 °C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A 2 and 15-deoxy-Δ12,14-prostaglandin J2. Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO2 and LNO2, showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pKa of target thiols increased. Increasing concentrations of the detergent octyl-β-D-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids.

Original languageEnglish (US)
Pages (from-to)31085-31093
Number of pages9
JournalJournal of Biological Chemistry
Volume282
Issue number42
DOIs
StatePublished - Oct 19 2007
Externally publishedYes

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Addition reactions
Alkylation
Sulfhydryl Compounds
Glutathione
Cysteine
Fatty Acids
Kinetics
Rate constants
Proteins
Prostaglandins A
Nitration
Membrane structures
Spectrophotometry
Linoleic Acid
Oleic Acid
Detergents
Hydrogen Peroxide
Reaction rates
Anions
Nitric Oxide

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Baker, L. M. S., Baker, P. R. S., Golin-Bisello, F., Schopfer, F. J., Fink, M., Woodcock, S. R., ... Freeman, B. A. (2007). Nitro-fatty acid reaction with glutathione and cysteine: Kinetic analysis of thiol alkylation by a Michael addition reaction. Journal of Biological Chemistry, 282(42), 31085-31093. https://doi.org/10.1074/jbc.M704085200

Nitro-fatty acid reaction with glutathione and cysteine : Kinetic analysis of thiol alkylation by a Michael addition reaction. / Baker, Laura M.S.; Baker, Paul R.S.; Golin-Bisello, Franca; Schopfer, Francisco J.; Fink, Mitchell; Woodcock, Steven R.; Branchaud, Bruce; Radi, Rafael; Freeman, Bruce A.

In: Journal of Biological Chemistry, Vol. 282, No. 42, 19.10.2007, p. 31085-31093.

Research output: Contribution to journalArticle

Baker, LMS, Baker, PRS, Golin-Bisello, F, Schopfer, FJ, Fink, M, Woodcock, SR, Branchaud, B, Radi, R & Freeman, BA 2007, 'Nitro-fatty acid reaction with glutathione and cysteine: Kinetic analysis of thiol alkylation by a Michael addition reaction', Journal of Biological Chemistry, vol. 282, no. 42, pp. 31085-31093. https://doi.org/10.1074/jbc.M704085200
Baker, Laura M.S. ; Baker, Paul R.S. ; Golin-Bisello, Franca ; Schopfer, Francisco J. ; Fink, Mitchell ; Woodcock, Steven R. ; Branchaud, Bruce ; Radi, Rafael ; Freeman, Bruce A. / Nitro-fatty acid reaction with glutathione and cysteine : Kinetic analysis of thiol alkylation by a Michael addition reaction. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 42. pp. 31085-31093.
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AU - Baker, Paul R.S.

AU - Golin-Bisello, Franca

AU - Schopfer, Francisco J.

AU - Fink, Mitchell

AU - Woodcock, Steven R.

AU - Branchaud, Bruce

AU - Radi, Rafael

AU - Freeman, Bruce A.

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N2 - Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO2) and linoleic acid (LNO2) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO2 and LNO2 reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M-1s-1 and 355 M-1s -1, respectively, at pH 7.4 and 37 °C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A 2 and 15-deoxy-Δ12,14-prostaglandin J2. Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO2 and LNO2, showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pKa of target thiols increased. Increasing concentrations of the detergent octyl-β-D-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids.

AB - Fatty acid nitration by nitric oxide-derived species yields electrophilic products that adduct protein thiols, inducing changes in protein function and distribution. Nitro-fatty acid adducts of protein and reduced glutathione (GSH) are detected in healthy human blood. Kinetic and mass spectrometric analyses reveal that nitroalkene derivatives of oleic acid (OA-NO2) and linoleic acid (LNO2) rapidly react with GSH and Cys via Michael addition reaction. Rates of OA-NO2 and LNO2 reaction with GSH, determined via stopped flow spectrophotometry, displayed second-order rate constants of 183 M-1s-1 and 355 M-1s -1, respectively, at pH 7.4 and 37 °C. These reaction rates are significantly greater than those for GSH reaction with hydrogen peroxide and non-nitrated electrophilic fatty acids including 8-iso-prostaglandin A 2 and 15-deoxy-Δ12,14-prostaglandin J2. Increasing reaction pH from 7.4 to 8.9 enhanced apparent second-order rate constants for the thiol reaction with OA-NO2 and LNO2, showing dependence on the thiolate anion of GSH for reactivity. Rates of nitroalkene reaction with thiols decreased as the pKa of target thiols increased. Increasing concentrations of the detergent octyl-β-D-glucopyranoside decreased rates of nitroalkene reaction with GSH, indicating that the organization of nitro-fatty acids into micellar or membrane structures can limit Michael reactivity with more polar nucleophilic targets. In aggregate, these results reveal that the reversible adduction of thiols by nitro-fatty acids is a mechanism for reversible post-translational regulation of protein function by nitro-fatty acids.

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