INACTIVATION OF MOUSE-LIVER GLUTATHIONE-S-TRANSFERASE YFYF (PI CLASS)BY ETHACRYNIC-ACID AND 5,5'-DITHIOBIS-(2-NITROBENZOIC ACID)

Mouse liver glutathione S-transferase YfYf (Pi class) reacts with [C-1 4]ethacrynic acid to form a covalent adduct with a stoichiometry of 1 mol per mol of subunit. Proteolytic digestion of the enzyme-[C-14]etha crynic acid adduct with V8 protease produced an 11 kDa fragment contai ning radioactivity. Sequencing revealed this to be an N-terminal pepti de (minus the first 15 residues, terminating at Glu-112) which contain s only one cysteine residue (Cys-47). This is tentatively identified a s the site of ethacrynic attachment. Kinetic studies reveal that gluta thione S-conjugates protect against inactivation by ethacrynic acid, b ut the level of protection is not consistent with their potency as pro duct inhibitors. A model is proposed in which glutathione S-conjugates and ethacrynic acid compete for the free enzyme, and a second molecul e of ethacrynic acid reacts covalently with the enzyme-ethacrynic acid complex. The native protein contains one thiol reactive with 5,5'-dit hiobis-(2-nitrobenzoic acid) at neutral pH. The resultant mixed disulp hide, like the ethacrynic acid adduct, is inactive, but treatment with cyanide (which incorporates on a mol for mol basis) restores activity to 35% of that of the native enzyme.