Mixed disulfide with glutathione as an intermediate in the reaction catalyzed by glutathione reductase from yeast and as a major form of the enzyme in the cell

Glutathione reductase catalyzes the reduction of glutathione disulfide by N ADPH. The ran of the reductase is reduced by NADPH, and reducing equivalent s are passed to a redox-active disulfide to complete the first half-reactio n. The nascent dithiol of two-electron reduced enzyme (EH2) interchanges wi th glutathione disulfide forming two molecules of glutathione in the second half-reaction. It has long been assumed that a mixed disulfide (MDS) betwe en one of the nascent thiols and glutathione is an intermediate in this rea ction. In addition to the nascent dithiol composed of Cys(45) and Cys(50), th, enzyme contains an acid catalyst, His(456), having a pK(a) of 9.2 that protonates the first glutathione (residue numbers refer to the yeast enzyme sequence). Reduction of yeast glutathione reductase by glutathione and reo xidation of EH2 by glutathione disulfide indicate that the mixed disulfide accumulates, in particular, at low pH. The reaction of glutathione disulfid e with EH2 is stoichiometric in the absence of an excess of glutathione. Th e equilibrium position among E-ox, MDS, and EH2 is determined by the glutat hione concentration and is not markedly influenced by pH between 6.2 and 8. 5. The mixed disulfide is the principal product in the reaction of glutathi one with oxidized enzyme (E-ox) at pH 6.2. Its spectrum can be distinguishe d from that of EH2 by a slightly lower thiolate (Cys(50))-FAD charge-transf er absorbance at 540 nm. The high GSH/GSSG ratio in the cytoplasm dictates that the mixed disulfide will be the major enzyme species.