Redox equilibria between the regulatory thiols of light/dark-modulated chloroplast enzymes and dithiothreitol: fine-tuning by metabolites.
Three light/dark-modulated chloroplast enzymes, namely NADP-dependent malate dehydrogenase (EC 1.1.1.82), D-fructose 1,6-bisphosphatase (EC 3.1.3.11), and phosphoribulokinase (EC 2.7.1.19) were purified to apparent homogeneity from spinach leaves. Equilibrium constants for the covalent modification of the regulatory disulfide bonds of these enzymes in dithiothreitol (DTT)-redox buffer were determined according to a previously published method in the literature (Clancey and Gilbert (1987) J. Biol. Chem. 262, 13545-13549). The thiol/disulfide-redox potential (Kox) was defined as the ratio of reduced to oxidized dithiothreitol at which 50% of the maximal enzyme activity was observed after equilibrium had been established. All Kox values were very high, comparable to those of extracellular disulfide containing proteins: 0.23 +/- 0.02 for NADP-malate dehydrogenase, 0.59 +/- 0.17 for phosphoribulokinase, and 0.70 +/- 0.16 for D-fructose 1,6-bisphosphatase. The equilibrium constants for the reactions between these enzymes and the redox buffers were also determined in the presence of various concentrations of specific metabolites known to influence the rates of reduction and oxidation. Increasing concentrations of D-fructose 1,6-bisphosphate in the presence of Ca2+ shift the equilibrium constant between D-fructose 1,6-bisphosphatase and the DTT-redox buffer to much lower values. A decreasing NADPH/(NADP + NADPH) ratio increases the Kox of NADP-malate dehydrogenase in the redox buffer to very high values. For PRK, low concentrations of ATP result in a slight decrease of the Kox that is not further affected by higher ATP concentrations. The differences of the equilibrium constants of NADP-malate dehydrogenase and D-fructose 1,6-bisphosphatase as dependent upon the NADPH/(NADP + NADPH) ratio and the concentration of D-fructose 1,6-bisphosphate, respectively, reflect a mechanism of feed-back and feed-forward regulation by the product NADP and the substrate D-fructose 1,6-bisphosphate, respectively. Thus the actual activation state of these two key enzymes of chloroplast metabolism are determined in an independent manner. The relatively small effect of the ATP concentration upon the redox potential of phosphoribulokinase indicates that fine-regulation at this step might be achieved on another level (e.g., catalysis or aggregation state).
Faske
M
M
Universität Osnabrück, FRG.
Holtgrefe
S
S
Ocheretina
O
O
Meister
M
M
Backhausen
J E
JE
Scheibe
R
R
D002736:Chloroplasts / Q000201:enzymology*; D004229:Dithiothreitol; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D008027:Light; D008291:Malate Dehydrogenase / Q000378:metabolism; D050538:Malate Dehydrogenase (NADP+); D010084:Oxidation-Reduction; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D006597:Fructose-Bisphosphatase; D004229:Dithiothreitol