Carbonyl cyanide phenylhydrazones as probes of the anionic activator site of the human erythrocyte glutathione adduct transport ATPase.

We have previously shown that the ATPase activity associated with the erythrocyte glutathione adduct transporter is also stimulated by 2,4-dinitrophenol and p-trifluoromethoxy carbonylcyanide phenylhydrazone, both well-known anionic and lipophilic uncouplers of oxidative phosphorylation by mitochondria [C. G. Winter, D. C. DeLuca, and H. Szumilo (1994) Arch. Biochem. Biophys. 314, 17-22]. In this paper, we report the testing of a series of ring-substituted carbonylcyanide phenylhydrazones as activators of the ATPase. All of the compounds tested stimulated the ATPase to similar extents, based on Vmax values. The K0.5 for stimulation of the ATPase depended on the electron-withdrawing characteristics of the ring substituents, resulting in a Hammett linear free energy relationship for the m- and p-substituted derivatives. The slope of this relationship, with lower K0.5 values for electron-withdrawing substituents, suggests that an anionic residue in the active site partially discourages binding of this class of activators. ortho-Substituted carbonylcyanide phenylhydrazones do not follow this relationship, but show lower apparent affinities than expected from their pKa values. This finding suggests that steric effects in that region of the binding site negatively influence the affinity.