RESUMEN
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'.
Asunto(s)
Glutatión Reductasa/metabolismo , Tirosina/metabolismo , Sustitución de Aminoácidos/genética , Sitios de Unión , Catálisis , Transporte de Electrón , Activación Enzimática/genética , Polarización de Fluorescencia , Glutatión/análogos & derivados , Glutatión/farmacología , Glutatión Reductasa/antagonistas & inhibidores , Glutatión Reductasa/genética , Humanos , Leucina/genética , NADP/metabolismo , Oxidación-Reducción , Serina/genética , Espectrometría de Fluorescencia , Tirosina/genéticaRESUMEN
A series of newly synthesized N(10)-arylisoalloxazines--some of which are known to be antimalarial agents--were studied as inhibitors of human glutathione reductase (GR;NADPH + GSSG + H(+) <==> NADP(+) + 2GSH). The flavoenzyme was inhibited with IC(50) values between - 1 and 100 microM in the presence of 100 microM NADPH. The isoalloxazines and N(3)-methylisoalloxazines with a 4'-chlorophenyl or a 3',5'-dichlorophenyl group at N10 were found to be the most promising inhibitors of GR, although even the bulkier 10-naphthyl and -anthryl derivatives were also effective inhibitors. In contrast, at position N3 of the isoalloxazine ring, the size of the substituent was found to strongly influence the inhibitory effect. Introduction of a carboxymethyl group at N3--which markedly increased the solubility of the derivative in aqueous solutions-- caused a rise in the IC(50) values by 1 order of magnitude. 8-Fluoro- and 8-azido-10-arylisoalloxazines were potent inhibitors of GR; consequently position C8 of the benzenoid subnucleus instead of N3 should be considered for introducing substituents. No correlation was observed between the inhibitory strength of several isoalloxazines and their redox potential as measured by cyclovoltammetry. The crystallographic analysis of GR complexed with 10-(4'-chlorophenyl)-3-(carboxymethyl)isoalloxazine and 10-(3',5'-dichlorophenyl)-3-(carboxymethyl)isoalloxazine, respectively, revealed the presence of one inhibitor molecule bound at the 2-fold axis of the homodimeric protein. This location is consistent with fluorescence titration measurements and enzyme kinetic studies in solution which gave no indication for binding at the substrate sites.