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PMID: 17720974 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

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

The Journal of biological chemistry ·Vol. 282 ·No. 42 ·2007-10-19 ·Pages 31085-93

Baker LM, Baker PR, Golin-Bisello F, Schopfer FJ, Fink M, Woodcock SR, Branchaud BP, Radi R, Freeman BA

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(-1)S(-1) and 355 M(-1)S(-1), respectively, at pH 7.4 and 37 degrees 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 A2 and 15-deoxy-Delta(12,14)-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-beta-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.

MeSH Terms
Cysteine/chemistry Fatty Acids, Unsaturated/chemistry Glutathione/chemistry Kinetics Nitric Oxide/chemistry Nitro Compounds/chemistry Oxidation-Reduction Sulfhydryl Compounds/chemistry
Chemicals
Fatty Acids, Unsaturated Nitro Compounds Sulfhydryl Compounds Nitric Oxide Glutathione Cysteine
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Baker Laura M S
Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Baker Paul R S
Golin-Bisello Franca
Schopfer Francisco J
Fink Mitchell
Woodcock Steven R
Branchaud Bruce P
Radi Rafael
Freeman Bruce A
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Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
0021-9258
DOI
10.1074/jbc.M704085200
Published
2007-10-19
Epub
2007-00-25
Pages
31085-93
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC2169496
Subset
IM
Grants
NHLBI NIH HHS · R37 HL058115 · United States
NHLBI NIH HHS · R01 HL058115 · United States
NHLBI NIH HHS · R01 HL058115-10 · United States
NHLBI NIH HHS · HL64937 · United States
NHLBI NIH HHS · R01 HL064937 · United States
NHLBI NIH HHS · HL58115 · United States
NHLBI NIH HHS · R01 HL064937-07 · United States
External Links
PubMed source DOI: 10.1074/jbc.M704085200 PMC Full Text View journal details
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