CHARACTERIZATION OF CHICKEN-LIVER GLUTATHIONE-S-TRANSFERASE (GST) A1-1 AND A2-2 ISOENZYMES AND THEIR SITE-DIRECTED MUTANTS HETEROLOGOUSLY EXPRESSED IN ESCHERICHIA-COLI - IDENTIFICATION OF LYS-15 AND SER-208 ONCGSTA1-1 AS RESIDUES INTERACTING WITH ETHACRYNIC-ACID

Escherichia coli-expressed chicken-liver glutathione S-transferase, cG STA1-1, displays high ethacrynic acid (EA)-conjugating activity. Molec ular modelling of cGSTA1-1 with EA in the substrate binding site revea ls that the side chain of Phe-lll protrudes into the substrate binding site and possibly interacts with EA. Replacement of Phe-lll with alan ine resulted in an enzyme (F111A mutant) with a 4.5-fold increase in E A-conjugating activity (9.2 mmol/min per mg), and an incremental Gibbs free energy (Delta Delta G) of 4.0 kJ/mol lower than that of the wild -type cGSTA1-1. Two other amino acid residues that possibly interact w ith EA are Ser-208 and Lys-15. Substitution of Ser-208 with methionine generated a cGSTA1-1(F111AS208M) double mutant that has low EA-conjug ating activity (2.0 mmol/min per mg) and an incremental Gibbs free ene rgy of +3.9 kJ/mol greater than the cGSTA1-1(F111A) single mutant. The cGSTA1-1(F111A) mutant, with an additional Lys-15-toleucine substitut ion, lost 90% of the EA-conjugating activity (0.55 mmol/min per mg). T he K-m values of the cGSTA1-1(F111A) and cGSTA1-1(F111AK15L) mutants f or EA are nearly identical. The wild-type cGSTA2-2 isoenzyme has a low EA-conjugating activity (0.56 mmol/min. per mg). The k(cat) of this r eaction can be increased 2.5-fold by substituting Arg-15 and Glu-104 w ith lysine and glycine respectively. The K-m(RA) Of th, cGSTA2-2(R15KE 104G) double mutant is nearly identical with that of the wild-type enz yme. Another double mutant, cGSTA2-2(E104GL208S), has a K-m(RA) that i s 3.3-fold lower and a k(cat) that is 1.8-fold higher than that of the wild-type enzyme. These results, taken together, illustrate the inter actions of Lys-15 and Ser-208 on cGSTA1-1 with EA.