REDOX EQUILIBRIA BETWEEN THE REGULATORY THIOLS OF LIGHT DARK-MODULATED CHLOROPLAST ENZYMES AND DITHIOTHREITOL - FINE-TUNING BY METABOLITES

Autor(en): FASKE, M
HOLTGREFE, S
OCHERETINA, O
MEISTER, M
BACKHAUSEN, JE
SCHEIBE, R 
Stichwörter: Biochemistry & Molecular Biology; Biophysics; CHLOROPLAST ENZYME; FRUCTOSE-1,6-BISPHOSPHATASE; FRUCTOSE-BISPHOSPHATASE; LIGHT/DARK MODULATION; METABOLITE EFFECT; NADP-MALATE DEHYDROGENASE; NADP-MALATE-DEHYDROGENASE; PHOSPHORIBULOKINASE; PHOTOSYNTHESIS; PROTEIN; PURIFICATION; QUANTITATION; REDOX EQUILIBRIUM; REDUCTIVE ACTIVATION; RIBULOSE-5-PHOSPHATE KINASE; SPINACH
Erscheinungsdatum: 1995
Herausgeber: ELSEVIER SCIENCE BV
Journal: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY
Volumen: 1247
Ausgabe: 1
Startseite: 135
Seitenende: 142
Zusammenfassung: 
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 (K-ox) 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 K-ox 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 K-ox 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 K-ox 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).
ISSN: 01674838
DOI: 10.1016/0167-4838(94)00203-S

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