No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Carbon dioxide assimilation
Abstract
This chapter discusses the carbon dioxide assimilation in plants. All oxygenic (oxygen-evolving) organisms from the simplest prokaryotic cyanobacteria to the most complicated land plants have a common pathway for the reduction of CO2 to sugar phosphates. This pathway is known as the reductive pentose phosphate (RPP), Calvin-Benson or C3 cycle. Although the RPP cycle is the fundamental carboxylating mechanism, a number of plants have evolved adaptations in which CO2 is first fixed by a supplementary pathway and then released in the cells in which the RPP cycle operates. One of these supplementary pathways, the C4 pathway, involves special leaf anatomy and a division of biochemical labor between cell types. Plants endowed with this pathway, through greater efficiency, are able to flourish under conditions of high light intensity and elevated temperatures. A second supplementary pathway was first found in species of the Crassulaceae and is called Crassulacean acid metabolism (CAM). These plants are often found in dry areas and fix CO2 at night into C4 acids. During the day, the leaves can close their stomata to conserve water, while CO2 released from the C4 acids is converted to sugar phosphates by the RPP cycle using absorbed light energy. CO2 fixation is also found in many bacteria, both photosynthetic and non-photosynthetic. The purple sulfur and purple nonsulfur bacteria employ the RPP cycle as do plants. The photosynthetic green bacteria, however, use a group of ferredoxin-linked carboxylases in a pathway known as the reductive carboxylic acid cycle.
... It is a key enzyme in gluconeogensis and photosynthesis [5]. This enzyme is part of the C 4 -dicarboxylic acid cycle, for the fixation of atmospheric CO 2 in mesophyll cells and subsequent transport (as malate) to bundle sheath cells for photosynthesis [6][7][8]. The result of Wang et al. indicated that the two enzymes known to limit C 4 photosynthesis, increase of PPDK, not Rubisco content, corresponds to the recovery and maintenance of photosynthetic capacity [9]. ...
... Lipids are more energy dense, which have frequently been applied as fuel feedstocks. The former studies indicated the feasibility of enhancing photosynthesis through overexpression of PPDK gene [5][6][7][8][9][10][11][12]. ...
Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of AMP, phosphoenolpyruvate (PEP) and pyrophosphate (PPi) to ATP, pyruvate and inorganic phosphate (Pi). It is a key enzyme in gluconeogensis and photosynthesis that is responsible for reversing the reaction performed by pyruvate kinase in Embden-Meyerhof-Parnas glycolysis. A cDNA clone for the Dunaliella parva PPDK was isolated by sequencing. Then the 3'-RACE and 5'-cDNA amplification were conducted based on the obtained sequence. The molecular characterization of the PPDK gene was described. The Dunaliella parva PPDK gene cDNA sequence was 3249 bp, which contained 2595 bp coding region and 654 bp 3'-untranslated regions. The deduced amino acid sequence of Dunaliella parva PPDK showed significant homology to the known PPDK from Volvox carteri and Chlamydomonas reinhardtii. This study provided foundation for further research on the function analysis and overexpression of PPDK genes. To our knowledge this is the first reported.
... In contrast, in photosynthetic organisms, PPDK primarily works in the gluconeogenic direction converting pyruvate, P i and ATP to phosphoenolpyruvate (PEP), PP i and AMP. In tropical plants, this enzyme is part of the C 4 -dicarboxylic acid cycle, for the fixation of atmospheric CO 2 in mesophyll cells (which are in contact with air) and subsequent transport (as malate) to bundle sheath cells, for photosynthesis [11,12]. Recently, the fortuitous cloning of a glycosomal PPDK from Trypanosoma brucei and demonstration of enzyme activity in a range of other trypanosomatids has raised important questions as to its physiological role in these organisms that also possess cytosolic pyruvate kinase [13]. ...
... removal of PEP or hydrolysis of PP i by pyrophosphatase readily drives the reaction in the reverse direction. Thus, it can function in ATP synthesis in organisms such as C. symbiosum and E. histolytica [10,32 -34] that lack a classical pyruvate kinase, whereas in Propionibacterium shermanii and C 4 plants PPDK substitutes for pyruvate carboxylase and PEP carboxykinase in PEP formation [11,32]. PEP is important in a number of anaplerotic reactions (gluconeogenesis, CO 2 fixation in photosynthesis and maintaining tricarboxylic acid cycle intermediates in the mitochondrion), as well as in the biosynthesis of aminophosphonates, sialic acids, and aromatic amino acids via the shikimic acid pathway. ...
We have cloned and characterised a gene that encodes a putative pyruvate phosphate dikinase (PPDK) from Trypanosoma cruzi, an enzyme that catalyses the reversible conversion of phosphoenolpyruvate to pyruvate. PPDK is absent in mammalian cells, but has been found in a wide variety of other organisms, including plants and bacteria. In T. cruzi, two genes (PPDK1 and PPDK2) are present in a tandem array localised on a 1 Mbp chromosome. Northern and Western blot analyses indicates that PPDK is expressed as a 100-kDa protein in epimastigote, amastigote and trypomastigote forms. PPDK1 and PPDK2 encode an identical protein of 100.8 kDa with a C-terminal extension ending with the sequence AKL, a signal for glycosomal import. Both T. cruzi and T. brucei enzymes possess a 23-residue insertion, that is absent in other PPDKs. A three-dimensional alignment with the crystal structure of the enzyme from Clostridium symbiosum predicts that this insertion is located on the surface of the nucleotide-binding domain. Phylogenetic studies indicate that bacterial and protist PPDKs cluster as a separate group from those of plants. The evolutionary implications and possible role of this enzyme in T. cruzi is discussed.
This chapter aims to discuss those biochemical interconversions involved in monosaccharide biosynthesis (anabolism) and degradation (catabolism). In particular, emphasis will center on defining the major pathways of monosaccharide metabolism and their interactions while outlining current knowledge on the molecular properties of relevant enzyme-catalyzed reactions involved in these processes.
The turnover time of enzymes, when saturated with their substrates, may be of the order of 10−5 to 0.1 s. This time is taken up by the need of the protein to undergo changes in conformation. In the cell, however, enzymes are virtually never saturated with substrate, indeed the concentration of an enzyme may exceed that of its substrate. The rate of metabolic processes is strongly dependent on diffusion, so that the actual turnover times of enzymes may be measured in seconds, and complete pathways may take 10–100 s to adjust to changes in the supply of feedstock.
The activities of enzymes involved in carbon dioxide fixation in photosynthetic organisms are regulated by a variety of physiological factors such as substrate and effector levels, intracellular pH and ion concentration, and redox control (1). Redox control can be effected through NADP+/NADPH ratios, glutathione, or thioredoxin. Thioredoxin is a small disulfide-containing protein which has been implicated in the regulation of a number of processes in plant metabolism, including carbon dioxide fixation, sulfate metabolism, and deoxyribonucleotide synthesis. Two thioredoxin fractions occur in chloroplasts of higher plants which can modulate different enzymes although with some overlap of specificities. Thioredoxin in photosynthetic organisms can in turn be reduced by either ferredoxin or NADPH. Like higher plant chloroplasts, two dissimilar thioredoxins occur in the cyanobacterium, Anabaena sp. strain PCC 7120. The cyanobacteria provide a simple model system for investigating processes occurring in the chloroplast.
The relationship between photosynthesis and nitrogen assimilation in chloroplasts and cells of C3-plants is reviewed. The spatial organization of these processes, the distribution of energy among them, and the mechanisms of their interaction are considered. Special attention is paid to the control mechanisms involving the compartmentation of processes, partitioning of metabolites, and transport of substrates and products.
In recent years the C4 pathway of carbon dioxide fixation in plants has been associated with specific anatomical, physiological, and biochemical characteristics (Hatch et al., 1971; Black, 1973). Grasses exhibiting this pathway have become known as C4 grasses and they are restricted to the sub-families Panicoideae and Eragrostoideae of the family Gramineae (Poaceae) (Smith and Brown, 1973). Although C4 grasses occur in areas from the hot-wet tropics to the cool temperate their greatest concentration is in the semi-arid tropics and sub-tropics, which are characterised by high solar radiation, high daytime temperatures and evaporative demand, and frequent diurnal and seasonal water stress (Hartley, 1958a, b; Hartley and Slater, 1960; Coaldrake, 1964; Cooper, 1970).
Photosynthesis is largely to do with energy transduction; the conversion of light energy into electrical energy into chemical energy. Precisely how much light energy is needed to bring about the reduction of one molecule of carbon dioxide and the release of one molecule of oxygen (the quantum requirement) is a matter of fundamental importance and one which has attracted much past controversy. This article concludes that a minimum quantum requirement of eight, as demanded by the Z‐scheme, is obviously consistent with much contemporary work which puts the measured value for C 3 leaves close to nine. Moreover, while values of less than eight (obtained in some circumstances with micro‐organisms), are a reminder that nothing is beyond challenge they are not, in the absence of confirmation and extension, sufficiently compelling to demand rejection of either the Z‐scheme or current measuring procedures. This article also shows why, even if the underlying minimum requirement was now accepted beyond all reasonable doubt, there would still be very good reasons for continuing, indefinitely, to measure actual photosynthetic efficiency in the natural environment. It discusses some of the implications of the fact that all plants, if not stressed, appear to photosynthesize at the same rate in low light. It explains the role of fluorescence in its relation to quantum yield, the possibility that the rate of photosynthesis might be determined from fluorescence measurements alone, and that a combination of fluorescence and gas exchange measurements could provide new information about the manner in which ‘dark respiration’ is affected by light. It indicates how contemporary interest in all of these matters has focused attention on the necessity for safe dissipation of excitation energy by leaves and on the manner by which this might be achieved.
CONTENTS
Summary
325
I.
Excitation
325
II.
Quantum requirement
326
III.
Learning from fluorescence
335
IV.
Safely dissipated
340
Acknowledgements
342
References
342
The phosphoribulokinase, when it is in a reduced state in a bi-enzyme complex, is more active than when it is oxidized. This complex dissociates upon dilution to give a metastable reduced form of phosphoribulokinase, which differs from the stable form isolated beside the complex. The kinetic parameters of the reduced stable phosphoribulokinase and those of the complex are very similar, unlike those of the metastable form. Although the kinetic mechanism of the reduced stable form is ordered, with ribulose-5-phosphate binding first, ATP binds first to the phosphoribulokinase in the complex and to the metastable form. Therefore, phosphoribulokinase bears an imprint from glyceraldehyde-3-phosphate dehydrogenase after disruption of the complex. Dissociation of phosphoribulokinase from the complex also enhances its flexibility. The imprinting and greater flexibility result in the catalytic constant of dissociated phosphoribulokinase being 10-fold higher than that of the enzyme in the complex.
Imprinting corresponds to stabilization-destabilization energies resulting from conformation changes generated by protein-protein interactions. The energy stored within the metastable phosphoribulokinase is mainly used to decrease the energy barrier to catalysis.
Extracts have been rapidly prepared from spinach leaves, and the activity of fructose-6-phosphate, 2-kinase (Fru6P,2kinase) and fructose-2,6-bisphosphatase (Fru2.6P 2 ase) measured. The enzyme activities do not change during light-dark transitions, but there is an increase of Fru6P,2- kinase and decrease of Fru2,6P 2 ase activity over several hours in the light. This increase of the Fru6P,2kinase: Fru2,6P 2 :ase ratio shows that a previously unrecognized mechanism, which may include protein modification, controls Fru2,6P 2 levels in leaves. It operates as sucrose accumulates in the leaf, and will be involved in regulating the partitioning of photosynthate.
Various aspects of the biochemistry of photosynthetic carbon assimilation in C3 plants are integrated into a form compatible with studies of gas exchange in leaves. These aspects include the kinetic properties of ribulose bisphosphate carboxylase-oxygenase; the requirements of the photosynthetic carbon reduction and photorespiratory carbon oxidation cycles for reduced pyridine nucleotides; the dependence of electron transport on photon flux and the presence of a temperature dependent upper limit to electron transport. The measurements of gas exchange with which the model outputs may be compared include those of the temperature and partial pressure of CO2(p(CO2)) dependencies of quantum yield, the variation of compensation point with temperature and partial pressure of O2(p(O2)), the dependence of net CO2 assimilation rate on p(CO2) and irradiance, and the influence of p(CO2) and irradiance on the temperature dependence of assimilation rate.
The chloroplastic NADP-dependent malate-dehydrogenase (EC 1.1.1.82) activity is modulated by light and dark. The enzyme is activated upon illumination of intact or broken chloroplasts or by incubation with dithiothreitol, whereas dark has the opposite effect. The present communication shows an additional regulation of the light modulation: in isolated intact pea chloroplasts, light activation was inhibited in the presence of electron acceptors such as sodium bicarbonate, 3-phosphoglycerate or oxaloacetate, which consume NADPH2 and produce NADP. With broken chloroplasts, addition of NADP resulted in a pronounced lag phase of NADP-dependent malate dehydrogenase light activation, while NADPH2 was without any effect. The extent of the lag phase was correlated to the amount of NADP added. When light was replaced by dithiotreitol, the inhibition effect was even more pronounced. It was assumed that NADP inhibits the modulation reaction directly: reduced thioredoxin, a potent mediator of activation by light, or dithiotreitol appear to counteract NADP in a competitive manner. The results indicate a physiological role of NADP in the regulation of chloroplastic NADP-dependent malate dehydrogenase which is capable of removing electrons from the chloroplast, via oxaloacetate reduction and malate export. Thus an NADP concentration sufficient for continuous photosynthetic electron flow may be achieved.
Sap extracted from attached leaves of two-to three-week-old maize plants witt the aid of a roller device was almost devoid of bundle-sheath contamination as judged by the distribution of mesophyll and bundle-sheath markers. The extraction could be done very rapidly (less than 1 s) and the extract immediately quenched in HClO4 or reserved for enzyme assay. Comparison of the contents of metabolites in intact leaves and in the leaf extract allowed estimation of the distribution of metabolites between the bundle-sheath and the mesophyll compartments. Substantial amounts of metabolites such as malate and amino acids were present in the non-photosynthetic cells of the midrib. In the illuminated leaf, triose phosphate was predominantly located outside the bundle-sheath while the major part of the 3-phosphoglycerate was in the bundle sheath. The results indicate the existence of concentration gradients of triose phosphate and 3-phosphoglycerate in the leaf which are capable of maintaining carbon flow between the mesophyll and bundle-sheath cells during photosynthesis. There was no evidence for the existence of a gradient of pyruvate between the bundle-sheath and the mesophyll cells.
Chloroplast fructose-1,6-bisphosphatase hysteresis in response to modifiers was uncovered by carrying out the enzyme assays in two consecutive steps. The activity of chloroplast fructose-1,6-bisphosphatase, assayed at low concentrations of both fructose-1,6-bisphosphatase and Mg2+, was enhanced by preincubating the enzyme with dithiothreitol, thioredoxin f, fructose 1,6-bisphosphate, and Ca2+. In the time-dependent activation process, fructose 1,6-bisphosphate and Ca2+ could be replaced by other sugar biphosphates and Mn2+, respectively. Once activated, chloroplast fructose-1,6-bisphosphatase hydrolyzed fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate in the presence of Mg2+, Mn2+, or Fe2+. The A0.5 for fructose 1,6-bisphosphate (activator) was lowered by reduced thioredoxin f and remained unchanged when Mg2+ was varied during the assay of activity. On the contrary, the S0.5 for fructose 1,6-bisphosphate (substrate) was unaffected by reduced thioredoxin f and depended on the concentration of Mg2+. Ca2+ played a dual role on the activity of chloroplast fructose-1,6-bisphosphatase; it was a component of the concerted activation and an inhibitor in the catalytic step. Provided dithiothreitol was present, the activating effectors were not required to maintain the enzyme in the active form. Considered together these results strongly suggest that the regulation of fructose-1,6-bisphosphatase in chloroplast occurs at two different levels, the activation of the enzyme and the catalysis.
Pure phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from liver is strongly inhibited by ATP, whereas crude phosphofructokinase is only slightly inhibited by ATP. A factor that is removed from the enzyme during purification and can prevent the inhibition of phosphofructokinase by ATP has been isolated. The factor can be resolved into three components that differ in molecular weights, as shown by gel filtration on Sephadex G-25. These factors overcome the ATP inhibition but have no effect on the catalytic activity under the optimum assay conditions. Furthermore, AMP acts syngeristically with the activation factor in reversing ATP inhibition. It is proposed that the activation of phosphofructokinase by the activation factor and AMP is sufficient to account for the glycolytic flux in the liver.
The mechanism of activation of thioredoxin-linked NADP-malate dehydrogenase was investigated by using 14C-iodoacetate and 14C-dansylated thioredoxin m, and Sepharose affinity columns (thioredoxin m, NADP-malate dehydrogenase) as probes to monitor enzyme sulfhydryl status and enzyme-thioredoxin interaction. The data indicate that NADP-malate dehydrogenase, purified to homogeneity from corn leaves, is activated by a net transfer of reducing equivalents from thioredoxin m, reduced by dithiothreitol, to enzyme disulfide groups, thereby yielding oxidized thioredoxin m and reduced enzyme. The appearance of new sulfhydryl groups that accompanies the activation of NADP-malate dehydrogenase appears to involve a structural change that is independent of the formation of a stable complex between the enzyme and reduced thioredoxin m. The data are consistent with the conclusion that oxygen promotes deactivation of NADP-malate dehydrogenase through oxidation of SH groups on reduced thioredoxin and on the reduced (activated) enzyme.
The carboxylation of ribulose 1,5-bisphosphate (RuBP) by the enzyme RuBP carboxylase is the primary CO2 fixing reaction of photosynthesis. It is therefore reasonable to assume that any factor which changes the rate of photosynthesis must ultimately do so through an effect on this reaction. The reaction rate may be influenced by the concentration of the two substrates (CO2 and RuBP), the activity of RuBP carboxylase present, or by inhibitors of the reaction. For example, limitation of photosynthesis by CO2 is easily determined based on the response of photosynthetic rate to CO2 concentration in the intercellular spaces. It is clear that photosynthesis is limited by CO2 at CO2 concentrations around ambient, and this observation is consistent with kinetic data for RuBP carboxylase and CO2in
vitro.
Robert Hill’s [1] first isolation of chloroplasts opened the door to the study of in vitro photosynthesis on a major scale and much of our present understanding of electron, transport, photophosphorylation and transport of metabolites between the chloroplast and the cellular environment derives from work on thylakoids and intact chloroplasts. In pursuing this approach ourselves we have been conscious for many years of the need to relate “in vitvo information” to the physiology of the cell and the leaf.
The photochemical apparatus affects the activity of chloroplastic and of cytoplasmic enzymes in several ways. Light-mediated activation or inactivation results in changes in maximal velocity of several enzymes. Within the chloroplast the lightmodulated enzymes and other enzymes are also affected by pH and Mg2+ and/or by ATP and NADPH. Photochemistry then affects stromal chemistry at two levels: (1) by altering the catalytic properties of several enzymes; (2) through a superimposed regulation by effectors and substrates generated by the photochemical apparatus.
Photosynthesis in C3 plants is usually inhibited by air levels of O2. Because C3 plants have no accessory metabolic pathways for concentrating CO2, the characteristics of photosynthesis such as oxygen sensitivity, measured with intact leaves can be readily interpreted at the biochemical level. This paper will discuss the biochemical basis for O2 sensitivity of C3 photosynthesis, the predictions of O2 sensitivity which follow from known mechanisms, and the metabolic information which O2 sensitivity measurements can give.
Following demonstrations that malic acid synthesis in crassulacean acid metabolism (CAM) plants at night requires only the carboxylation of phosphoenolpyruvate, glycolysis has been presumed to supply this substrate. This study has examined the path of carbon in CAM plants at night to establish the involvement of intermediary pools of metabolites in other processes, and to define characteristics of the pathway which are likely to be involved in its regulation. In both Bryophyllum tubiflorum Barv. and Kalanchoe daigremontiana Hamet et Perrier, carbon loss from starch was insufficient to account for malic acid gains. Quantitative examination of the pool sizes of various carbohydrates and malic acid during the dark showed that starch was only part of a larger glucan pool which was the carbon source material for phosphoenolpyruvate supply. Observations of the utilization of 14C-labelled glucan in a CAM plant in the dark supported the role of this compound as primary carbon donor for acid synthesis. The mainstream of carbon flow was shown not to pass through the free sugar pools, and it was also proposed that malic and citric acids were able to exchange carbon via tricarboxylic acid cycle reactions. Examination of the influence of temperature on the glucan-malic acid relationship suggested that the depressed malic acid levels accumulated after nights at higher temperature were due to regulation of the reactions concerned with acid synthesis from glucan.
Enzymes involved in the movement of carbon from glucan to malic acid in the crassulacean acid metabolism (CAM) plant, Kalanchoe daigremontiana were assayed. The kinetic characteristics determined for the enzymes from this plant were similar to those already known for the same enzymes from non-CAM tissue. °-Amylase activity could not be demonstrated in the CAM leaf and glucokinase activity was low. These results, together with a high level of phosphorylase, suggested that the latter enzyme was involved in trasfer of glucan breakdown products to glycolysis. The activity of pyruvate kinase was only 1.7% of the activity of phosphoenolpyruvate (PEP) carboxylase, suggesting that pyruvate production from PEP at night posed little drain on PEP supply for malic acid synthesis. Starch losses and glycolytic enzyme activities of non-CAM plants were sufficient to allow dark acidification comparable to that of CAM plants.
Ribulosebisphosphate carboxylase/oxygenase (EC 4.1.1.39) (rubisco) must be fully activated in order to catalyze the maximum rates of photosynthesis observed in plants. Activation of the isolated enzyme occurs spontaneously, but conditions required to observe full activation are inconsistent with those known to occur in illuminated chloroplasts. Genetic studies with a nutant of Arabidopsis thaliana incapable of activating rubisco linked two chloroplast polypeptides to the activation process in vivo. Using a reconstituted light activation system, it was possible to demonstrate the participation of a chloroplast protein in rubisco activation. These results indicate that a specific chloroplast enzyme, rubisco activase, catalyzes the activation of rubisco in vivo.
The metabolite levels in the mesophyll of leaves of Zea mays L. have been compared with the regulatory properties of the cytosolic fructose-1,6-bisphosphatase from the mesophyll to show how withdrawal of triose phosphate for sucrose synthesis is reconciled with generation of the high concentrations of triose phosphate which are needed to allow intercellular diffusion of carbon during photosynthesis. i) A new technique is presented for measuring the intercellular distribution of metabolites in maize. The bundle-sheath and mesophyll tissues are partially separated by differential homogenization and filtration through nylon nets under liquid nitrogen. ii) considerable gradients of 3-phosphoglycerate, triose phosphate, malate and phosphoenolpyruvate exist between the mesophyll and bundle sheath which would allow intercellular shuttles to be driven by diffusion. These gradients could result from the distribution of electron transport and the Calvin cycle in maize leaves. iii) consequently, the mesophyll contains high concentrations of triose phosphate and fructose-1,6-bisphosphate. iv) Most of the regulator metabolite fructose-2,6-bisphosphate, is present in the mesophyll. v) The cytosolic fructose-1,6-bisphosphatase has a lower substrate affinity than that found for the enzyme from C3 species, especially in the presence of inhibitors like fructose-2,6-bisphosphate. vi) This lowered affinity for substrate makes it possible to reconcile use of triose phosphate for sucrose synthesis with the maintenance of the high concentration of triose phosphate in the mesophyll needed for operation of photosynthesis in this species.
The incorporation of (14)C into sucrose and hexose phosphates during steady-state photosynthesis was examined in intact leaves of Zea mays L. plants. The compartmentation of sucrose synthesis between the bundle sheath and mesophyll cells was determined by the rapid fractionation of the mesophyll and comparison of the labelled sucrose in this compartment with that in a complete leaf after homogenisation. From these experiments it was concluded that the majority of sucrose synthesis occurred in the mesophyll cell type (almost 100% when the time-course of sucrose synthesis was extrapolated to the time of (14)C-pulsing). The distribution of enzymes involved in sucrose synthesis between the two cell types indicated that sucrose-phosphate synthetase was predominantly located in the mesophyll, as was cytosolic (neutral) fructose-1,6-bisphosphatase activity. Stromal (alkaline) fructose-1,6-bisphosphatase activity was found almost exclusively in the bundle-sheath cells. No starch was found in the mesophyll tissue. These data indicate that in Zea mays starch and sucrose synthesis are spatially, separated with sucrose synthesis occurring in the mesophyll compartment and starch synthesis in the bundle sheath.
Activities catalyzing the synthesis of fructose-2,6-bisphosphate (fructose-6-phosphate,2-kinase or Fru-6-P,2K) and its breakdown (fructose-2,6-bisphosphatase or Fru-2,6-P2ase) were identified in leaves of corn (Zea mays), a C4 plant. Fru-6-P,2K and Fru-2,6-P2ase were both localized mainly, if not entirely, in the leaf mesophyll cells. A partially purified preparation containing the two activities revealed that the kinase and phosphatase were regulated by metabolite effectors in a manner generally similar to their counterparts in C3 species. Thus, corn Fru-6-P,2K was activated by inorganic phosphate (Pi) and fructose-6-phosphate, and was inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fru-2,6-P2ase was inhibited by its products, fructose-6-phosphate and Pi. However, unlike its spinach equivalent, corn Fru-2,6-P2ase was also inhibited by 3-phosphoglycerate and, less effectively, by dihydroxyacetone phosphate. The C4 Fru-6-P,2K and Fru-2,6-P2ase were also quite sensitive to inhibition by phosphoenolpyruvate, and each enzyme was also selectively inhibited by certain other metabolites.
Conditions required for the reductive activation of purified, spinach chloroplast fructose-1,6-bisphosphatase (EC 3.1.3.11) have been determined in vitro. Full reductive activation was observed only when fructose-1,6-bisphosphate and Mg²⁺ were present at the same time as the reducing agent (dithiothreitol). Reduction in the absence either of fructose-1,6-bisphosphate or of Mg²⁺ slowly and irreversibly inactivated the enzyme. The concentration of fructose-1,6-bisphosphate that must be present during reduction for maximum activation depends upon the divalent cation present: it is highest with Mg²⁺, lower with Ca²⁺, and lowest when both Mg²⁺ and Ca²⁺ are present. A scheme for the reductive activation and inactivation of the enzyme is presented.
Treatment of carrot roots with ethylene led to: (a) a doubling of the fructose-2,6-bisphosphate content; (b) a general increase in the concentration of glycolytic intermediates; and (c) an increase in the extractable activity of fructose-6-phosphate,2-kinase, the enzyme synthesizing fructose-2,6-bisphosphate from fructose-6-phosphate and adenosine triphosphate.
Initial ribulose 1,5-biphosphate carboxylase activity (that present in vivo) and total activity (that which is made available after complete activation by CO2 and Mg2+ were measured in extracts of field-grown soybean (Glycine max cv. Will) leaves collected over a diurnal period. Initial activity increased with increasing irradiance, saturating at 1.2 mmol m−2 s−1, and decreased when irradiance fell below this level. Total activity increased about 3-fold during the first 4 h and remained constant for the rest of the period. Initial activity as a percentage of total activity was about 50% at the beginning and end of the diurnal period and remained near 100% for 6 h in the middle of the day. Leaf photosynthesis and percent activation both increased similarly with increasing irradiance. The lower total activity observed in the morning hours could not be increased by gel-filtration of the extracts in the presence of Mg2+ and CO2 concentrations favoring complete activation. No marked changes were observed in the amount of total leaf protein on a dry weight basis in leaf samples collected throughout the diurnal period. We propose that light has two effects on ribulose 1,5-biphosphate carboxylase activity: (1) light facilitates activation by CO2 and Mg2+, and (2) promotes the conversion of inactive enzyme present after a prolonged dark period to a form which is able to be activated.
A specific transport process, known as the phosphate translocator, makes it possible for the products of photosynthesis to be exported from the chloroplast to the plant cell. Its appearance during evolution was a key event for the development of eukaryotic plants.
The effect of stromal metabolites on the light-activated form of ribulose-5-phosphate kinase was studied with the enzyme rapidly extracted from illuminated spinach chlorplasts. In some instances, the effect of metabolites on the dark-inactivated enzyme extracted from darkened chloroplasts was also investigated. (1) The light-activated form of the enzyme is competitively inhibited with respect to ribulose 5-phosphate by 6-phosphogluconate, ribulose 1,5-bisphosphate, 3-phosphoglycerate and phosphate. Also, fructose 1,6-bisphosphate is inhibitory. All these compounds, except ribulose 1,5-bisphosphate, show an increasing inhibitory effect at lower pH values. Therefore, in the presence of these inhibitors, ribulose-5-phosphate kinase becomes strongly pH dependent. These compounds also exert an inhibitory effect on the dark-inactivated enzyme. (2) The assay of stromal levels of 6-phosphogluconate showed that this compound increased dramatically during a light-dark transient. (3) The dark-inactivated form of ribulose-5-phosphate kinase is strongly inhibited by ADP, the inhibition being competitive with respect to ATP. (4) A simulation of stromal metabolite levels in the enzyme activity assay indicates that in illuminated chloroplasts ribulose-5-phosphate kinase attains only about 4% of its maximal activity. When the fully light-activated enzyme is assayed under conditions occurring in the stroma in the dark, the activity is further decreased by a factor of 20. The same assay with the dark-inactivated enzyme yields an activity of virtually zero. (5) These results demonstrate that in the chloroplasts ribulose-5-phosphate kinase can not only be very efficiently switched off in the dark, but also be subjected to fine control during the illuminated state through the action of stromal metabolites.
(1) The amino-acid analysis of homogeneous NADP: malate dehydrogenase (l-malate: NADP+ oxidoreductase, E.C. 1.1.1.82) from pea leaves revealed the presence of three half-crystine residues per subunit Mr 38500). The determination of the total thiol groups of the denatured and reduced enzyme which was performed by incorporation of as well as by measuring the reaction with 5,5′ dithiobis(2-nitrobenzoic acid) spectrophotochemically were in full agreement with this finding. (2) It could be established that upon activation of NADP: malate dehydrogenase two thiol groups per subunit are formed. The third thiol is apparently not available in the native enzyme. The kinetics of thiol group formation and of expression of catalytic activity suggested that the fully reduced tetramer is the only species exhibiting NADP: malate dehydrogenase activity. (3) The inhibition of activation of NADP: malate dehydrogenase by NADP could be explained in terms of the bound NADP preventing the reduction of the regulatory disulfide bridge
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
To understand how a steady-state flux through a pathway can be maintained and how it can be changed from one rate to another, knowledge of the kinetic structure of the pathway is essential. The kinetic structure of a pathway is presented and its application to the experimental approach in metabolic control, the influence of hormones and the relationship between hormones and secondary messengers are discussed.
Fructose 2,6-bisphosphate, a regulatory metabolite discovered in animal cells, plays a central role in regulating carbon metabolism in leaves.
Cell-free preparations of the cyanobacterium (bluegreen alga) Nostoc muscorum were assayed for thioredoxins and enzymes catalyzing the ferredoxin and NADP-linked reduction of thioredoxin. Nostoc was found to have two different thioredoxins: one of approximate molecular weight 16,000 (designated Nostoc thioredoxin f) that selectively activated chloroplast fructose 1,6-bisphosphatase, and another of approximate molecular weight 9,000 (designated Nostoc thioredoxin m) that selcetively activated chloroplast NADP-malate dehydrogenase. The two thioredoxins could be reduced either chemically with dithiothreitol or photochemically with ferredoxin and ferredoxin-thioredoxin reductase which, like the recently found regulatory iron-sulfur protein ferralterin, was present in Nostoc cells. Nostoc ferredoxin-thioredoxin reductase appeared to be similar to its chloroplast counterpart in enzyme specificity, molecular weight, and spectral properties. The Nostoc and spinach chloroplast ferredoxin-thioredoxin reductases as well as their thioredoxins, ferredoxins, and chlorophyll containing membranes were interchangeable in activating chloroplast fructose 1,6-bisphosphatase and NADP-malate dehydrogenase. There was no evidence for an NADP-linked thioredoxin reductase such as that of E. coli. The results are in accord with the conclusion that the cyanobacteria resemble higher plants in having a functional ferredoxin/thioredoxin system rather than an NADP/thioredoxin system typical of other bacteria.
The standard physiological free energy changes of reactions of glycolysis, the reductive pentose phosphate cycle (photosynthetic carbon reduction cycle) and the oxidative pentose phosphate cycle have been calculated from available data. The concentrations of metabolites of the photosynthetic carbon reduction cycle, measured during steady-state photosynthesis in Chlorella pyrenoidosa in the presence of radioactive tracers, and the concentrations of some intermediates of the oxidative pentose phosphate cycle measured during a subsequent dark period, have been employed to calculate the free energy changes of each reaction of the reductive cycle and of some of the reactions of the oxidative cycle during steady-state light and dark conditions. With respect to the magnitude of the negative free energy change, at steady state, such reactions have been found to be of two types. Those with high negative free energy changes (−6 to −11 kcal) are in each case reactions from which there exists independent evidence of a role in metabolic regulation. Those with small negative free energy changes (o to −2 kcal) are not regulated reactions and are highly reversible. Thus most of the negative free energy change occurring under steady-state conditions in this metabolic system is dissipated for purposes of control.
The uptake of phosphate and phosphorylated compounds into the chloroplast stroma has been studied by silicone layer filtering centrifugation. 1. Inorganic phosphate, 3-phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde phosphate are transported across the envelope leading to an accumulation in the chloroplast stroma. This uptake proceeds by a counter exchange with phosphate and phosphorylated compounds present there. 2. The transport shows saturation characteristics allowing the determination of Km and V. 3. The phosphorylated compounds transported act as competitive inhibitors of the transport. From measurements of the Km and Ki the specificity of the transport is described. 4. The transport of inorganic phosphate and 3-phosphoglycerate is inhibited by p-chloromercuriphenyl sulfonate, pyridoxal 5'-phosphate and trinitrobenzene sulfonate. 5. The activation energy of phosphate transport as determined from the temperature dependence is evaluated to be 16 kcal (0--12 degrees C). 6. It is concluded that inorganic phosphate, 3-phosphoglycerate, dihydroxy-acetone phosphate and glyceraldehyde phosphate are transported by the same carrier, which has been nominated phosphate translocator. 7. Simultaneous measurements of the proton concentration in the medium and the transport into the chloroplasts show that the transfer of 3-phosphoglycerate involves a transfer of a proton into the same direction. 8. Measurements of the pH dependence of the transport indicate that all substances including 3-phosphoglycerate are transported by the phosphate translocator as divalent anions. 9. The physiological function of the phosphate translocator is discussed.
Recent studies have shown that the light-dark mediated regulation of the leaf photosynthetic enzyme pyruvate, Pi dikinase results from interconversion between an active nonphosphorylated form of the enzyme and an inactive form phosphorylated on a threonine residue. These phosphorylation and dephosphorylation reactions are apparently catalyzed by a single protein termed the pyruvate, Pi dikinase regulatory protein and, notably, both reactions are mechanistically unique. We consider the evidence that this regulatory protein belongs to a group of unusual bifunctional enzymes that catalyze opposing reactions, apparently at separate catalytic sites on the same polypeptide. In three of the four known cases these bifunctional enzymes interconvert the active and inactive forms of another enzyme. The possible advantages of such opposing reactions being catalyzed by the same protein are considered.
The complexation of ribulosebiphosphate carboxylase with CO2, Mg2+, and carboxyarabinitol bisphosphate (CABP) to produce the quaternary enzyme-carbamate-Mg2+-CABP complex closely mimics the formation of the catalytically competent enzyme-carbamate-Mg2+-3-keto-CABP form during enzymatic catalysis. Quaternary complexes were prepared with various metals (Mg2+, Cd2+, Mn2+, Co2+, and Ni2+) and with specifically 13C-enriched ligands. 31P and 13C NMR studies of these complexes demonstrate that the activator CO2 site (carbamate site), the metal binding site, and the substrate binding site are contiguous. It follows that both the carboxylase and oxygenase activities of this bifunctional enzyme are influenced by the structures of the catalytic and activation sites.
Using size-exclusion high-performance liquid chromatography, it is shown that phosphoenolpyruvate carboxylase from Crassula argentea, a crassulacean acid metabolism (CAM) plant, exists primarily in the form of a tetramer of a 100-kDa subunit at night and as a dimer of the same subunit during the day. The tetrameric enzyme from night leaves is not inhibited by malate, while the dimeric form from day leaves can be completely inhibited by malate. The purified day, or dimer, form of the enzyme can be converted to the tetramer by concentration and exposure to Mg2+. When thus converted, the tetramer is insensitive to malate inhibition, and is more strongly activated by glucose 6-phosphate than the dimer. The purified night, or tetramer, form is converted to the dimer by incubation for 60 min at pH 8.2. This enzyme may also be converted to the dimer by adding 1.5 mM malate to the elution buffer, but preincubation for 15 min with phosphoenolpyruvate prevents disaggregation when chromatographed with buffer containing malate. Preincubation with 1mM EDTA and subsequent chromatography with buffer containing malate shows a progressive dissociation of the tetrameric form with increasing time of preincubation. The implications of these observations for the diurnal regulation of phosphoenolpyruvate carboxylase in CAM metabolism are discussed.
The identities of the products of the ribulose diphosphate oxygenase reaction were confirmed by combined gas chromatography and mass spectrometry to be phosphoglycolate and 3-phosphoglycerate. Oxygen-18, supplied as molecular oxygen, was incorporated into one of the carboxyl oxygen atoms of phosphoglycolate. No label appeared in 3-phosphoglycerate, the other reaction product. When the reaction was carried out in a medium containing [18O]-water, the carboxyl groups of both products were singly labeled. A reaction mechanism is proposed. Cyanide inhibited the ribulose diphosphate oxygenase reaction in a manner consistent with the formation of an inactive enzyme-cyanide-substrate complex. Neither catalase nor superoxide dismutase (erythrocuprein) had any effect on the oxygenation reaction. In contrast to previous reports, purified preparations of spinach leaf fraction-1 protein were found to contain less than 14% of the copper required for a stoichiometry of one atom of copper per molecule of enzyme.
The standard physiological free energy changes of reactions of glycolysis, the reductive pentose phosphate cycle (photosynthetic carbon reduction cycle) and the oxidative pentose phosphate cycle have been calculated from available data. The concentrations of metabolites of the photosynthetic carbon reduction cycle, measured during steady-state photosynthesis in Chlorella pyrenoidosa in the presence of radioactive tracers, and the concentrations of some intermediates of the oxidative pentose phosphate cycle measured during a subsequent dark period, have been employed to calculate the free energy changes of each reaction of the reductive cycle and of some of the reactions of the oxidative cycle during steady-state light and dark conditions. With respect to the magnitude of the negative free energy change, at steady state, such reactions have been found to be of two types. Those with high negative free energy changes (-6 to -11 kcal) are in each case reactions from which there exists independent evidence of a role in metabolic regulation. Those with small negative free energy changes (o to -2 kcal) are not regulated reactions and are highly reversible. Thus most of the negative free energy change occurring under steady-state conditions in this metabolic system is dissipated for purposes of control. By the criterion of negative free energy change, ribulosediphosphate carboxylase, and fructose- and heptosediphosphatases are regulated enzymes. The activities of these enzymes are known to be high in the light and low in the dark. Phosphoribulokinase, which mediates the one reaction with an intermediate negative free energy change (-3.82 kcal) also may be a regulated enzyme with greater activity in the light than in the dark. In the oxidative cycle, the reaction mediated by glucose-6-phosphate dehydrogenase has a very high negative free energy change and appears to be active in the dark and inactive in the light. One function of these controls is thought to be the exclusive operation of the reductive cycle in the light and the oxidative cycle in the dark.
1.1. Leaves of an albino mutant and of normal green seedlings of maize have high levels of readily extractable alkaline inorganic pyrophosphatase (EC 3.6.1.1) when grown under strong illumination. About one-tenth as much activity is found in both types of plants when grown in the absence of light. A brief illumination of dark-grown seedlings induces a large increase in readily extractable pyrophosphatase activity in the subsequent dark period.2.2. In light-grown maize the enzyme is localized in the chloroplasts but is readily washed out by aqueous solvents. The enzyme is highly specific for Mg2+ and PPi, and has optimal activity between pH 8 and 9. Leaf extracts of sorghum have similar high activity; many other plants, and other maize tissues have much less activity.
Chloroplast fructose-1,6-bisphosphatase, isolated from spinach leaves, was activated by preincubation with Ca2+ (or Mn2+), fructose-1,6-bisphosphate and dithiothreitol-reduced thioredoxin-f. Upon activation, the enzyme displayed high activity when measured at low concentrations of both fructose-1,6-bisphosphate and Mg2+. On the contrary, the activity of chloroplast fructose-1,6-bisphosphatase was inhibited by Ca2+. These results suggest that Ca2+ (or Mn2+) is potentially important in the regulation of the chloroplast fructose-1,6-bisphosphatase reaction (activation and catalysis).
We have developed a method for the concomitant purification of several components of the ferredoxin/thioredoxin system of spinach chloroplasts. By applying this method to spinach-leaf extract or spinach-chloroplast extract we separated and purified three thioredoxins indigenous to chloroplasts. The three thioredoxins, when reduced, will activate certain chloroplast enzymes such as fructose-1,6-bisphosphatase and NADP-dependent malate dehydrogenase. Fructose- 1,6-bisphosphatase is activated by thioredoxin f exclusively. Malate dehydrogenase is activated by thioredoxin mb and thioredoxin mb in a similar way, and it is also activated by thioredoxin f but with different kinetics.
All three thioredoxins have very similar relative molecular masses of about 12000 but distinct isoelectric points of 6.1 (thioredoxin f), 5.2 (thioredoxin mb) and 5.0 (thioredoxin mc). The amino acid composition as well as the C-terminal and N-terminal sequences have been determined for each thioredoxin. Thioredoxin f exhibits clear differences in amino acid composition and terminal sequences when compared with the m-type thioredoxins. Thioredoxin mb and thioredoxin mc, however, are very similar, the only difference being an additional lysine residue at the N-terminus of thioredoxin mb.
Amino acid analyses, terminal sequences, immunological tests and the activation properties of the thioredoxins support our conclusion that thioredoxins mb and me are N-terminal redundant isomers coming from one gene whereas thioredoxin f is a different protein coded by a different gene.
The amino acid sequences surrounding the active sites of spinach chloroplast thioredoxins m and f have been determined. Both types of thioredoxins share common ancestor genes with the E. coli one, demonstrated by invariant active site sequences. The m-type thioredoxins have closer homology with the E. coli one in the sequence analyzed as well as in enzymatic specificity, whereas the f-type is less homologous both in sequence and specificity. It suggests that the m-type gene represents a prototype conserved throughout evolutionary processes whereas the f-type has undergone mutations resulting in a modified specificity.
The effects of pH, NaCl, and malate2- on the equilibrium between dimeric and higher-molecular-weight forms of NAD malic enzyme from Solanum tuberosum var. Chieftain have been analyzed by monitoring the kinetic changes associated with disaggregation [S. D. Grover and R. T. Wedding (1982) Plant Physiol. 70, 1169-1172]. At pH values above 7.0 the enzyme was disaggregated to the dimeric, high-Km(malate) form by preincubation with NaCl, with a half-maximal effect at 25 mM. At low pH the enzyme remained in the low-Km(malate) (tetramer or octamer) form. Malate protected against disaggregation to the high-Km form in preincubation, and this effect was half-maximal at 6 mM. At pH 7.3, in the absence of malate, half-maximal disaggregation occurred at 580 nM enzyme. Varying the enzyme concentration in the assay led to kinetic changes which fit equations based on an associating enzyme model [B. I. Kurganov (1967) Mol. Biol. (Moscow) 1, 17-27]. This analysis confirmed that the dimer has intrinsic activity, with Vm somewhat lower than that of the tetramer but a Km(malate) that was 9-fold higher than that of the tetramer. Malate decreased the Kd for disaggregation of the enzyme during assay approximately 20-fold, with a half-maximal effect at 3 to 4 mM. In contrast, high NaCl concentrations in the assay increased the Kd for disaggregation in a manner which was competitive with the effect of malate on Kd. The physiological significance of these aggregation state changes is discussed.
NADP-malate dehydrogenase was purified from leaves of Zea mays in the absence of thiol-reducing agents by (NH4)2SO4, polyethylene glycol, and pH fractionation followed by dye-ligand affinity chromatography and gel filtration. The purified enzyme is completely inactive (no activity detected between pH 6 and 9) but can be reactivated by thiol-reducing agents including dithiothreitol and thioredoxin. The active enzyme shows distinctly alkaline pH optima when assayed in either direction; Km values at pH 8.5 are oxaloacetate, 18 microM; malate, 24 mM; NADPH, 50 microM; and NADP, 45 microM. The reduction of oxaloacetate is inhibited by NADP (competitive with respect to NADPH, Ki = 50 microM). The molecular weight of the native inactive or active enzyme is 150,000 with subunits of Mr 38,000. Active enzyme is much more sensitive (greater than 50-fold) to heat denaturation than is the inactive enzyme and is irreversibly inactivated by N-ethylmaleimide whereas the inactive enzyme is insensitive to this reagent. The active and inactive forms of NADP-malate dehydrogenase are assumed to correspond to dithiol and disulfide forms of the enzyme, respectively. The relative coenzyme-binding affinities of inactive NADP-malate dehydrogenase differ by a factor of 10(2) from the binding affinities for active NADP-malate dehydrogenase and 10(4) for non-thiol-regulated NAD-specific malate dehydrogenase. It is proposed that the 100-fold change in differential binding of NADP and NADPH upon conversion of NADP-malate dehydrogenase to the disulfide form may sufficiently alter the equilibrium of the central enzyme-substrate complexes, and hence the catalytic efficiency of the enzyme, to explain the associated loss of activity.
In addition to its well-established function in supplying the energy for carbon dioxide assimilation, light plays a regulatory
role in photosynthesis. The ferredoxin/thioredoxin system is a major mechanism whereby light functions in this capacity. Here,
light absorbed by chlorophyll is converted via ferredoxin into a reductant messenger, reduced thioredoxin, that interacts
with key target enzymes, thereby changing their catalytic activities. In this way, the green plant achieves maximum efficiency
of its photosynthetic (light) and heterotrophic (dark) capabilities.