Glutathione Transferase Catalyzes the Addition of Glutathione to Nitro‐conjugated Linoleic Acid

Nitroalkene fatty acids are formed in vivo and are currently being tested in two Phase 2 clinical trials. They modulate cellular responses through Michael addition reactions with thiol‐containing proteins. Nitro‐conjugated linoleic acid (NO2‐CLA) is formed preferentially both in vivo and in vitro. There are two main positional isomers of NO2‐CLA, 9‐ and 12‐NO2‐CLA. They both react with low molecular weight thiols at two electrophilic sites located on the β‐ and δ‐carbons giving rise to reversible biphasic kinetics. The fast reaction corresponds to the formation of the β‐adduct (the kinetic product), while the slow reaction corresponds to the δ‐adduct (the thermodynamic product) (Turell, Vitturi, J. Biol. Chem., 2017). With the aim of further characterizing the biological chemistry of this bifunctional nitroalkene, we evaluated if its reaction with glutathione (GSH) is catalyzed by glutathione transferases (GST). The reaction was followed by monitoring NO2‐CLA decay at 330 nm using a stopped flow spectrophotometer. A mu‐type glutathione transferase from the parasitic platyhelminth Echinococcus granulosus (EgGST1) was used. Catalysis was observed in both phases when using a mixture of NO2‐CLA isomers. Remarkably, the δ‐addition was accelerated □13 times more than the β‐addition, 4.4 × 106 vs 3.4 × 105 M−1, respectively, at 2 mM GSH and 10 μM NO2‐CLA (25 °C, pH 7.4), suggesting specificity. To evaluate whether there is a preference of the enzyme for the NO2‐CLA isomer, purified 9‐ or 12‐ NO2‐CLA were used. No difference was observed between them. The reaction between GSH and nitro‐oleic acid (NO2‐OA), a nitroalkene fatty acid with only one electrophilic site in β, was also catalyzed by this enzyme. An acceleration of 1 × 105 M−1 was observed (2 mM GSH, 20 μM NO2‐OA), in line with the value obtained for the β‐addition of NO2‐CLA. We are now further characterizing this reaction which is expected to affect nitroalkene fatty acids metabolism and excretion and thus impact their signaling actions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.