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-d initrophenol and p-trifluoromethoxy carbonylcyanide phenylhydrazone, b oth well-known anionic and lipophilic uncouplers of oxidative phosphor ylation by mitochondria [C. G. Winter, D. C. DeLuca, and H. Szumilo (1 994) Arch. Biochem. Biophys. 314, 17-22]. In this paper, we report the testing of a series of ring-substituted carbonylcyanide phenylhydrazo nes as activators of the ATPase. All of the compounds tested stimulate d the ATPase to similar extents, based on V-max values. The K-0.5 for stimulation of the ATPase depended on the electron-withdrawing charact eristics of the ring substituents, resulting in a Hammett linear free energy relationship for the m- and p-substituted derivatives. The slop e of this relationship, with lower K-0.5 values for electron-withdrawi ng substituents, suggests that an anionic residue in the active site p artially discourages binding of this class of activators. ortho-Substi tuted carbonylcyanide phenylhydrazones do not follow this relationship , but show lower apparent affinities than expected from their pK(a) va lues. This finding suggests that steric effects in that region of the binding site negatively influence the affinity. (C) 1997 Academic Pres s.