The reaction mechanism of glutathione reductase from human erythrocytes

Abstract

1. 1. The reaction mechanism of glutathione reductase (NAD(P)H:oxidized glutathione oxidoreductase, EC 1.6.4.2) was studied. By varying the GSSG concentration, a series of parallel lines was obtained in the 1/vvs 1/[NADPH] plots. NADP+ is a competitive inhibitor towards NADPH and a noncompetitive one towards GSSG. Both GSSG and NADPH were inhibitory at high concentrations. In the 1/v vs. 1/[GSSG] plots only parallel lines were obtained at low NADPH concentrations. The maximum velocity was dependent on the Na+ concentration. 2. 2. With NADH as electron donor, a series of converging lines was obtained in the 1/v vs. 1/[NADH] plots; at high [GSSG] activation was observed. The maximum velocity was not affected by the Na+ concentration. NADH+ did not inhibit these reactions. 3. 3. The results were interpreted in terms of a combination of simplified ordered bi-bi and ping-pong bi-bi mechanism and individual rateconstants were calculated.Opposite effects of the Na+ concentration on the reaction rate of GSSG with the NADPH- and the NADH-reduced enzyme were found. 4. 4. The influence of the temperature on the velocity of the NADPH dependent reaction in relation to different concentrations of buffer and GSSG led to either linear or nonlinear Arrhenius plots.