Glutathione reductase from germinated peas

Abstract

The preparation and some properties of glutathione reductase, prepared from germinated peas, are described. Confirmation that the enzyme is a flavoprotein containing FAD as the prosthetic group was obtained. The kinetic results are consistent with the view that the enzyme exists in 2 different oxidative states, with 1 substrate (NADPH) reacting with the oxidized form, and the other (GSSG) with the reduced form of the enzyme. The addition to the apoenzyme of both FAD and either NADPH or NADH is required for the formation of a sulphydryl group on the active center. In the reduced state the enzyme is readily inactivated by compounds which react with sulphydryl groups. The integrity of this sulphydryl group is not essential for, or concerned with, the binding of FAD to the apoenzyme. The oxidized form of the sulphydryl group is not that of a disulphide, and the fact that GSH is not liberated on treatment with NADPH shows that a disulphide group of the type Enzyme.S.SG is not present, although such a group may be formed as an intermediate during the transfer of the 2 electrons from the FAD to GSSG. The inhibition of the oxidized enzyme by oxidizing agents such as mercuric oxide, and the reactivation of the enzyme so treated with ammonium sulphide but not with thiols, suggest that a lone S atom similar to that existing in thiourea is present. The observation that the yellow oxidized enzyme changes in color to pink when NADPH is added, and the effect of p-chloro-mercuribenzoate in modifying the spectrum of the reduced enzyme (especially that portion which has been ascribed to the formation of flavin semiquinones), suggest that the FAD exists in this state in the reduced enzyme. GSSG may be bound on a secondary site on the oxidized enzyme, and the GSSG so bound can react with the active sulphydryl group produced when the oxidized enzyme is reduced with NADPH. Many salts compete competitively with GSSG for this site, and this is 1 of the causes for the inhibition of the enzyme by these salts. Sodium iodide also inhibits by a specific effect on the sulphydryl group on the active center of the reduced enzyme. The evidence suggests that this group is oxidized to a disulphide. There is no evidence, as yet, to indicate whether this bond is formed between sulphydryl groups on the same molecule, or between those on adjacent molecules.