Chloroplast glucose-6-phosphate dehydrogenase: Km shift upon light modulation and reduction.
Illumination of intact chloroplasts and treatment of chloroplast stroma with dithiothreitol (DTT) both inactivate glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) to less than 10% apparent activity when assayed under standard conditions. Illumination of intact protoplasts and incubation of leaf extract with DTT inactivate about 25-35% of the total G6PDH activity. In the leaf extract, however, further loss of activity is observed if NADP is absent. Light- and DTT-inactivated chloroplast G6PDH can be reactivated by oxidation with sodium tetrathionate or the thiol oxidant diamide. Chloroplast G6PDH is as sensitive toward reductive enzyme modulation in a stromal extract as are other light/dark modulated enzymes, e.g., NADP-malate dehydrogenase. Also, glutathione, provided it is kept reduced, is sufficient to cause inactivation. Light- and DTT-induced inactivation are shown to be due to a Km shift with respect to glucose-6-phosphate (G6P) from 1 to 35 and 43 mM, respectively, and with respect to NADP from 10 to 50 microM without any significant change of the Vmax. NADPH competitively (NADP) inhibits the enzyme (Ki = 8 microM). Reactivation by oxidation can be explained by an enhanced affinity of the oxidized enzyme toward G6P and NADP. The pH optimum of the reduced enzyme is more in the alkaline region (pH 9-9.5) as compared to that of the oxidized form (pH 8.0). The presence of 30 mM phosphate causes a shift of 0.5 to 1.0 pH unit into the alkaline region for both forms.
Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, Federal Republic of Germany.
R
R
Scheibe
A
A
Geissler
K
K
Fickenscher
D002736:Chloroplasts / Q000201:enzymology*; D003624:Darkness; D007887:Fabaceae / Q000201:enzymology; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism* / Q000528:radiation effects; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D010084:Oxidation-Reduction; D010944:Plants / Q000201:enzymology*; D010946:Plants, Medicinal; D011523:Protoplasts / Q000201:enzymology
D005954:Glucosephosphate Dehydrogenase; D005978:Glutathione