Red cell glutathione reductase: Mechanism of action of inhibitors

Abstract

1. 1. The mechanism of action of 3 inhibitors of human red cell glutathione reductase (NADPH:oxidized glutathione oxidoreductase, EC 1.6.4.2), hexavalent chromium (Cr(VI)), N-ethylmaleimide, and 2,5-dinitrobenzoic acid was studied in relation to the two forms of the enzyme, an active form with flavin adenine dinucleotide (FAD) and an inactive form without FAD. 2. 2. N-Ethylmaleimide (1 mM optimal) inhibits glutathione reductase activity both in the active form and in the inactive form to the same extent in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH). The inhibition of the active form of glutathione reductase by N-ethylmaleimide is completely reversible, but the effect of N-ethylmaleimide on the inactive form is irreversible. 3. 3. Hexavalent chromium (0.5 mM optimal) inhibited almost irreversibly 90% of glutathione reductase activity in the active form associated with FAD in the presence of NADPH. However, hexavalent chromium appears to be incapable of inhibiting the inactive form of glutathione reductase without FAD. Sulfhydryl group(s) of the enzyme protein of glutathione reductase are essential for irreversible inhibition of the active form by Cr(VI). 4. 4. 2,5-Dinitrobenzoic acid (4 mM optimal) irreversibly inhibited about 50% of glutathione reductase activity in both the active and the inactive forms to the same extent. Sulfhydryl group(s) or FAD appears to be unnecessary for inhibition of the enzymatic activity. 2,5-Dinitrobenzoic acid may bind irreversibly to some structure(s) of the enzyme protein other than sulfhydryl radicals.