Studies on the glutathione-dehydroascorbate oxidoreductase (EC 1.8.5.1) from wheat flour

Glutathione (GSH) dehydrogenase was partially purified from Wheat flour after extraction, ammonium sulfate precipitation, and ionic-exchange chromatography on diethylaminoethyl (DEAE) Sepharose CL6B. Kinetic studies showed that the optimum pH was close to 7.5. The K-m values varied between 0.15 and 0.28 mM for dehydroascorbic acid (DHA) and between 1.8 and 0.62 mM for GSH when pH was varied from 5.5 to 7.5. The kinetic pattern was consistent with a sequential mechanism for the binding of GSH and DHA. NaCl is a competitive inhibitor with respect to GSH and is uncompetitive with respect to DHA, which suggests that the enzyme combines with DHA before it does with GSH. IsoDHA can replace DHA as hydrogen acceptor but with a K-m of 1.2 mM. gamma-Glu-cys was enzymically oxidized but much less efficiently than GSH (V-m = 47 nkat/mL and K-m = 5.5 mM compared to V-m = 362 nkat/mL and K-m = 1.8 mM for GSH), whereas cysteine and cys-gly were not substrates. In the presence of DHA, addition of cysteine and cys-gly to small amounts of GSH causes a large activation of the enzymatic formation of ascorbic acid suggesting coupled oxidation of these thiols.