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

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-Δ^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 pK_a 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.