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'.

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

Isoalloxazine derivatives such as 1 a-d are promising anti-aryl-3-carboxymethylisoalloxazines 2a-d, and the isomeric malarial agents which act as inhibitors of the antioxidant en-3-methyl-lO-(N-methylpyri&niumyl)isoalloxazine salts 3 and zyme glutathione reductase and possibly of other proteins. 4. In addition, for the purpose of photoaffinity labeling The molecular mechanism of the pharmacological effects has experiments, the 10-aryl-8-azido-3-methylisoalloxazine 5 not been studied in detail because compounds l a -d are was designed. The syntheses and characterizations of these poorly soluble in aqueous solutions of physiological pH. In new flavins as well as an alternative synthetic approach to the present study we introduce two new types of isoalloxaz-the known antimalarials la-d are described. ine derivatives with improved solubility properties: The 10-Falciparum malaria, one of the world's most devastating diseases, is caused by the multiplication of the protozoal parasite Plasmodium falciparum in human erythrocytes. The increasing resistance of the parasite to conventional drugs, such as chloroquine, has led to a demand for new and cheap chemotherapeutic agents.In 1988 Cowden et al."] reported on the antimalarial activity of new members of the class of l0-aryl-3-methylisoalloxazines (la-d). The therapeutic effect is assumed to be based, at least in part, on the inhibition of the antioxidant enzyme glutathione reductase (GR)L2l, which is present both in the parasite and in the host cell. GR protects the parasitized erythrocytes from oxidative stress [3] by (re)generating the tripeptide glutathione (GSH) from glutathione disulfide (GSSG) according to the equation NADPH + GSSG + H+ * NADP+ + 2 GSH. Detailed information about interactions of GR with 10-arylisoalloxazines would be very valuable for the development of even more effective and less toxic flavin-type antimalarials. Although erythrocyte GR is one of the best characterized enzymes with regard to geometry and mechanism of ~a t a l y s i s [~~~] , it has not been possible so far to localize the binding site(s) of the flavin-type inhibitors by difference Fourier X-ray structural analysis: Attempts to soak GR in saturated solutions of the most potent inhibitor l a (ICs0 = 1 FM) failed because of the extremely poor solubility in aqueous solvents and the photolability of this compound [za].