Molecular Properties of Chloroplastic Thioredoxin f and the Photoregulation of the Activity of Fructose 1,6-Bisphosphatase

Abstract

Thioredoxin f has been isolated and purified to homogeneity from spinach chloroplasts. The protein is a dimer which dissociates in two apparently identical halves when ionic strength is raised. The dimer has a molecular weight of 16000. In its oxidized state, chloroplastic thioredoxin f has no sulfhydryl group directly available, even after denaturation of the protein. When reduced by dithiothreitol, four -SH groups become available on the dimer: Taken together, these results show that there exists one disulfide bridge per monomer. Reduction by dithiothreitol results in the breaking of two disulfide bridges and the appearance of four sulfhydryl groups.Monomeric thioredoxin f does not result in a significant activation of fructose 1,6-bisphosphatase. Full activation is obtained in presence of the dimeric protein. This is understandable since activation of fructose bisphosphatase necessitates reduction of two disulfide bridges of the enzyme.Full activation of fructose bisphosphatase is obtained in the presence of either an excess of dimeric thioredoxin f, or in the presence of dithiothreitol. Moreover a large excess of oxidized thioredoxin deactivates fructose bisphosphatase. These results suggest that there exists an equilibrium rTh + oFruPzase + oTh + rFruPzase where rTh and oTh stand for reduced and oxidized thioredoxin, oFruPzase and rFruPlase for oxidized and reduced fructose bisphosphatase. Moreover, this equilibrium must be strongly shifted towards the left.NADPH in presence of NADP+-reductase may reduce thioredoxin, and conversely reduced thioredoxin may reduce NADP' if the reductase is present. These results together with the existence of the above equilibrium provides a tentative scheme of fructose bisphosphatase inactivation in vivo in the dark. When the light has been turned off, electrons are transferred from thioredoxin to NADP' via NADP+-reductase. Thioredoxin becomes oxidized and then deactivates fructose bisphosphatase.It is now firmly established that COz fixation by green plants is controlled by light [l -31. This control seems to be effected thanks to several factors, such as the level of substrates and cofactors [4], the local Hc and cation concentration [5] and the light-dependent activation of enzymes belonging to the reductive pentose phosphate cycle [6]. The most extensively studied of these enzymes is, without doubt, chloroplastic fructose 1,6-bisphosphatase [7 -91. This enzyme is a tetramer made up of apparently identical subunits. Dithiothreitol can mimic the effect of light and can activate the enzyme in the dark. It has recently been shown that this activation is due to the reduction of two strategic disulfide bridges of the tetrameric enzyme, followed by a conformation change of the protein that can be monitored by spectrofluorimetry [lo].

Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis

“…In teases also ap-these studies, we noted that the apparent mol wt of Kalanchoe )parent mol wt thioredoxin f varied with the ionic strength of the buffer. When (data not shown), suggesting the formation of a dimer similar to that described for thioredoxins of spinach under these conditions (26). Alternative explanations for this chromatographic behavior can be tendered, however.…”

Modification of the reactivity of spinach chloroplast thioredoxin f by site-directed mutagenesis

“…In vivo, reduced glutathione in the chloroplast might prevent the formation of dimers. A thioredoxin f dimer has been reported earlier, however, this dimer could be separated by high ionic strength which excludes the presence of a disulfide bond [9].…”

Modelling C3 photosynthesis: A sensitivity analysis of the photosynthetic carbon-reduction cycle

“…Many of the regulatory complexities of this enzyme have been simplified by assuming that there is a hyperbolic rather than a sigmoid relationship between enzyme activity and the concentration of fructose 1,5-bisphosphate (FBP; see review by Leegood et al 1985). Modification of the structure of FBPase by reduced thioredoxin (Soulie et al 1981) changes both the Km(FBP) and the maximum catalytic velocity (Charles Results) and Halliwell 1980). Although the enzyme is not fully activated at PPFD values that are largely saturating for the rate of photosynthesis (Leegood and Walker 1982), it is assumed that under the conditions of the current experiments, the proportion of active FBPase is relatively insensitive to changes in the concentrations of PCR-cycle intermediates.…”