AMINO-ACID RESIDUE-104 IN AN ALPHA-CLASS GLUTATHIONE-S-TRANSFERASE ISESSENTIAL FOR THE HIGH SELECTIVITY AND SPECIFICITY OF THE ENZYME FOR 4-HYDROXYNONENAL

Murine mGSTA4-4 is a glutathione S-transferase with high activity and specificity for products of lipid peroxidation, including the cytotoxi c 4-hydroxynonenal (4-HNE), Physiological relevance of this enzyme in the defense against effects of oxidative stress cad be inferred from t he above biochemical properties, and has been also directly demonstrat ed by us in vivo. The identification of residues responsible for the h igh activity toward 4-HNE is facilitated by the availability of X-ray crystal structures of mGSTA4-4 and of hGSTA1-1, a structurally related enzyme which lacks activity for 4-HNE. Residues likely to be involved in 4-HNE recognition were identified by molecular modeling. One such residue, M104, was mutated to E104, as present in hGSTA1-1, The result ing M104E mutant had unchanged catalytic properties toward the model s ubstrate 1-chloro-2,4-dintrobenzene. However, the KM of mGSTA4-4(M104E ) for 4-HNE was increased more than sevenfold, while the V-max for tha t substrate remained essentially unchanged. We conclude that M104 code termines the recognition and binding of 4-HNE to the active center of mGSTA4-4. (C) 1996 Academic Press, Inc.