ROLE OF ACTIVE-SITE TYROSINE RESIDUES IN CATALYSIS BY HUMAN GLUTATHIONE-REDUCTASE

Tyr114 and Tyr197 are highly conserved residues in the active site of human glutathione reductase, Tyr114 in the glutathione disulfide (GSSG ) binding site and Tyr197 in the NADPH site. Mutation of either residu e has profound effects on catalysis. Y197S and Y114L have 17% and 14% the activity of the wild-type enzyme, respectively. Mutation of Tyr197 , in the NADPH site, leads to a decrease in K-m for GSSG, and mutation of Tyr114, in the GSSG site, leads to a decrease in K-m for NADPH. Th is behavior is predicted for enzymes operating by a ping-pong mechanis m where both half-reactions partially limit turnover. Titration of the wild-type enzyme or Y114L with NADPH proceeds in two phases, E-ox to EH2 and EH2 to EH2-NADPH. In contrast, Y197S reacts monophasically, sh owing that excess NADPH fails to enhance the absorbance of the thiolat e-FAD charge-transfer complex, the predominant EH2 form of glutathione reductase. The reductive half-reactions of the wild-type enzyme and o f Y114L are similar; FAD reduction is fast (similar to 500 s(-1) at 4 degrees C) and thiolate-FAD charge-transfer complex formation has a ra te of 100 s(-1). In Y197S, these rates are only 78 and 5 s(-1), respec tively. The oxidative half-reaction, the rate of reoxidation of EH2 by GSSG, of the wild-type enzyme is approximately 4-fold faster than tha t of Y114L. These results are consistent with Tyr197 serving as a gate in the binding of NADPH, and they indicate that Tyr114 assists the ac id catalyst His467'.