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 residue 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 Km for GSSG, and mutation of Tyr114, in the GSSG site, leads to a decrease in Km for NADPH. This behavior is predicted for enzymes operating by a ping-pong mechanism where both half-reactions partially limit turnover. Titration of the wild-type enzyme or Y114L with NADPH proceeds in two phases, Eox to EH2 and EH2 to EH2-NADPH. In contrast, Y197S reacts monophasically, showing that excess NADPH fails to enhance the absorbance of the thiolate-FAD charge-transfer complex, the predominant EH2 form of glutathione reductase. The reductive half-reactions of the wild-type enzyme and of Y114L are similar; FAD reduction is fast (approximately 500 s-1 at 4 degreesC) and thiolate-FAD charge-transfer complex formation has a rate of 100 s-1. In Y197S, these rates are only 78 and 5 s-1, respectively. The oxidative half-reaction, the rate of reoxidation of EH2 by GSSG, of the wild-type enzyme is approximately 4-fold faster than that of Y114L. These results are consistent with Tyr197 serving as a gate in the binding of NADPH, and they indicate that Tyr114 assists the acid catalyst His467'.
Biochemie-Zentrum, Heidelberg University, Germany.
R L
RL
Krauth-Siegel
L D
LD
Arscott
A
A
Schönleben-Janas
R H
RH
Schirmer
C H
CH
Williams
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D004579:Electron Transport; D004789:Enzyme Activation / Q000235:genetics; D005454:Fluorescence Polarization; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007930:Leucine / Q000235:genetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D012694:Serine / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
C030190:S-(2,4-dinitrophenyl)glutathione; D014443:Tyrosine; D012694:Serine; D009249:NADP; D005980:Glutathione Reductase; D005978:Glutathione; D007930:Leucine