Ribulose-1,5-bisphosphate Concentrations Research Articles

Regulation of Rubisco activity by interaction with chloroplast metabolites.

Rubisco activity is highly regulated and frequently limits carbon assimilation in crop plants. In the chloroplast, various metabolites can inhibit or modulate Rubisco activity by binding to its catalytic or allosteric sites, but this regulation is complex and still poorly understood. Using rice Rubisco, we characterised the impact of various chloroplast metabolites which could interact with Rubisco and modulate its activity, including photorespiratory intermediates, carbohydrates, amino acids; as well as specific sugar-phosphates known to inhibit Rubisco activity - CABP (2-carboxy-d-arabinitol 1,5-bisphosphate) and CA1P (2-carboxy-d-arabinitol 1-phosphate) through in vitro enzymatic assays and molecular docking analysis. Most metabolites did not directly affect Rubisco in vitro activity under both saturating and limiting concentrations of Rubisco substrates, CO2 and RuBP (ribulose-1,5-bisphosphate). As expected, Rubisco activity was strongly inhibited in the presence of CABP and CA1P. High physiologically relevant concentrations of the carboxylation product 3-PGA (3-phosphoglyceric acid) decreased Rubisco activity by up to 30%. High concentrations of the photosynthetically derived hexose phosphates fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) slightly reduced Rubisco activity under limiting CO2 and RuBP concentrations. Biochemical measurements of the apparent Vmax and Km for CO2 and RuBP (at atmospheric O2 concentration) and docking interactions analysis suggest that CABP/CA1P and 3-PGA inhibit Rubisco activity by binding tightly and loosely, respectively, to its catalytic sites (i.e. competing with the substrate RuBP). These findings will aid the design and biochemical modelling of new strategies to improve the regulation of Rubisco activity and enhance the efficiency and sustainability of carbon assimilation in rice.

Light- and CO2-saturated photosynthesis: enhancement by oxygen

Oxygen may enhance CO2-saturated photosynthesis in intact leaves, which display the Warburg effect when illuminated at the current atmospheric level of CO2 and O2, of about 350 microl l(-1) and 21%, respectively. The magnitude of the stimulation depends on irradiance. The K(M)(O2) of the stimulation is 128 microM (10.6% O2). Maximum enhancement in wheat leaves is 6.1 and 5.3 micromol m(-2) s(-1 )under 27.9 and 18.7 mW cm(-2), respectively, corresponding to a 25-30% increase in the ribulose 1,5-bisphosphate (RuBP) turnover rate if compared with O2-free ambient gas phase. The stimulation appears in 5-10 s after a sharp increase in O2. In response to a decrease in O2, the new stabilized rate is reached in 5-7 min. The stimulation does not involve any increase in the activity of Rubisco. The effect correlates with increased concentration of RuBP. Oxygen enhances CO2-saturated photosynthesis by acting as a terminal electron acceptor in the photosynthetic electron transport. The magnitude of the effect may be adopted as an index of the pseudocyclic photophosphorylation in vivo.

Distribution of fallover in the carboxylase reaction and fallover-inducible sites among ribulose 1,5-bisphosphate carboxylase/oxygenases of photosynthetic organisms.

The biphasic reaction course, fallover, of carboxylation catalysed by ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been known as a characteristic of the enzyme from higher land plants. Fallover consists of hysteresis in the reaction seen during the initial several minutes and a very slow suicide inhibition by inhibitors formed from the substrate ribulose-1,5-bisphosphate (RuBP). This study examined the relationship between occurrence of fallover and non-catalytic RuBP-binding sites, and the putative hysteresis-inducible sites (Lys-21 and Lys-305 of the large subunit in spinach RuBisCO) amongst RuBisCOs of a wide variety of photosynthetic organisms. Fallover could be detected by following the course of the carboxylase reaction at 1 mM RuBP and the non-catalytic binding sites by alleviation of fallover at 5 mM RuBP. RuBisCO from Euglena gracilis showed the same linear reaction course at both RuBP concentrations, indicating an association between an absence of fallover and an absence of the non-catalytic binding sites. This was supported by the results of an equilibrium binding assay for this enzyme with a transition state analogue. Green macroalgae and non-green algae contained the plant-type, fallover enzyme. RuBisCOs from Conjugatae, Closterium ehrenbergii, Gonatozygon monotaenium and Netrium digitus, showed a much smaller decrease in activity at 1 mM RuBP than the spinach enzyme and the reaction courses of these enzymes at 5 mM RuBP were almost linear. RuBisCO of a primitive type Conjugatae, Mesotaenium caldariorum, showed the same linear course at both RuBP concentrations. Sequencing of rbcL of these organisms indicated that Lys-305 was changed into arginine with Lys-21 conserved.

Modeling of continuously and directly analyzed biphasic reaction courses of ribulose 1,5-bisphosphate carboxylase/oxygenase.

This paper aims at clarifying the cause of the time-dependent, partial loss of the activity during reaction (so-called fallover) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from plant sources. This was done by comparing the reaction courses calculated using the reaction models constructed here based on the present conflicting two ideas on fallover with directly measured courses obtained with RuBisCO purified from spinach leaves. Since the ordinary methods with 14CO2 and indicator enzymes were not adequate for analyzing the progress of fallover, we followed the reaction by measuring the change of the light absorbance of ribulose 1,5-bisphosphate (RuBP) at 280 nm. Direct measurements of the reaction course showed that RuBisCO lost its activity with a rate constant of 6.1 to 6.5 x 10(-3) s-1 at both 0.5 and 2 mM RuBP. The rate constant of the recovery of the enzyme to show the original fallover was determined as 1.2 to 1.3 x 10(-3) s-1 with RuBisCO that had just experienced fallover. These constants were used in the models. Calculation with a model assuming the binding of xylulose 1,5-bisphosphate (XuBP) to the catalytic sites of the enzyme as the cause of fallover and using the reported dissociation constant of XuBP in the binding and the reported rate of the formation of XuBP from RuBP gave a rather linear reaction course. The minimum requirements for the model to be valid were that the rate of XuBP formation was more than once for every 600 turnovers, the dissociation constant of XuBP for the catalytic sites was less than 0.1 nM, and the binding of XuBP to the sites showed a strong negative cooperativity. Inclusion of non-catalytic RuBP-binding sites in the model was essential to elucidate the course at higher RuBP concentrations. The model constructed assuming that hysteresis was the cause of fallover could calculate the measured reaction courses for the initial 20 min of reaction of both 0.5 and 2mM RuBP. The rate constants of the hysteretic conformational changes of the predicted enzyme forms to others were given. The direct measurement of the long-term reaction course revealed the two phases in the decay of the activity; fast decay for the initial several minutes and subsequent slow decrease. Although the fast decay could be predicted by the hysteresis model, the slower one required the participation of inhibition by XuBP. We reasoned from these results and the reported characteristics of the binding of other sugar phosphates to the catalytic sites that the initial fast decay of the activity in fallover was due to the hysteretic property of the enzyme and the slower phase of fallover was due to the inhibition of XuBP.

Subsaturating Ribulose-1,5-Bisphosphate Concentration Promotes Inactivation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) (Studies Using Continuous Substrate Addition in the Presence and Absence of Rubisco Activase).

We developed a continuous-addition method for maintaining subsaturating concentrations of ribulose-1,5-bisphosphate (RuBP) for several minutes, while simultaneously monitoring its consumption by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This method enabled us to observe the effects of subsaturating RuBP and CO2 concentrations on the activity of Rubisco during much longer periods than previously studied. At saturating CO2, the activity of the enzyme declined faster when RuBP was maintained at concentrations near its Km value than when RuBP was saturating. At saturating RuBP, activity declined faster at limiting than at saturating CO2, in accordance with previous observations. The most rapid decline in activity occurred when both CO2 and RuBP concentrations were subsaturating. The activity loss was accompanied by decarbamylation of the enzyme, even though the enzyme was maintained at the same CO2 concentration before and after exposure to RuBP. Rubisco activase ameliorated the decline in activity at subsaturating CO2 and RuBP concentrations. The results are consistent with a proposed mechanism for regulating the carbamylation of Rubisco, which postulates that Rubisco activase counteracts Rubisco's unfavorable carbamylation equilibrium in the presence of RuBP by accelerating, in an ATP-dependent manner, the release of RuBP from its complex with uncarbamylated sites.

Dependence of Photosynthesis of Sunflower and Maize Leaves on Phosphate Supply, Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activity, and Ribulose-1,5-Bisphosphate Pool Size

Sunflower (Helianthus annuus L. cv Asmer) and maize (Zea mays L. cv Eta) plants were grown under controlled environmental conditions with a nutrient solution containing 0, 0.5, or 10 millimolar inorganic phosphate. Phosphate-deficient leaves had lower photosynthetic rates at ambient and saturating CO(2) and much smaller carboxylation efficiencies than those of plants grown with ample phosphate. In addition, phosphate-deficient leaves contained smaller quantities of total soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) per unit area, although the relative proportions of these components remained unchanged. The specific activity of Rubisco (estimated in the crude extracts of leaves) was significantly reduced by phosphate deficiency in sunflower but not in maize. Thus, there was a strong dependence of carboxylation efficiency and CO(2)-saturated photosynthetic rate on Rubisco activity only in sunflower. Phosphate deficiency decreased the 3-phosphoglycerate and ribulose-1,5-bisphosphate (RuBP) contents of the leaf in both species. The ratio of 3-phosphoglycerate to RuBP decreased in sunflower but increased in maize with phosphate deficiency. The calculated concentrations of RuBP and RuBP-binding sites in the chloroplast stroma decreased markedly with phosphate deficiency. The ratio of the stromal concentration of RuBP to that of RuBP-binding sites decreased in sunflower but was not affected in maize with phosphate deficiency. We suggest that a decrease in this ratio made the RuBP-binding sites more vulnerable to blockage or inactivation by tight-binding metabolites/inhibitors, causing a decrease in the initial specific activity of Rubisco in the crude extract from phosphate-deficient sunflower leaves. However, the decrease in Rubisco specific activity was much less than the decrease in the RuBP content in the leaf and its concentration in the stroma. A large ratio of RuBP to RuBP-binding sites may have maintained the Rubisco-specific activity in phosphate-deficient maize leaves. We conclude that the effect of phosphate deficiency is more on RuBP regeneration than on Rubisco activity in both sunflower and maize.

Rubisco activity of scenedesmus ecornis: What is wrong with initial activity determination?

A procedure was devised to measure the initial and total Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities for the green microalga, Scenedesmus ecornis. Total Rubisco activities corresponded well with photosynthetic carbon assimilation rates. Initial activities ranged from 10 to 40% of the total activities and did not correlate with photosynthetic rates. Investigations into potential causes of the reduced initial activities yielded modest increases in percentage of the total activity. Values of K m for ribulose-1,5-bisphosphate (RuBP) were similar for both initial and CO 2-Mg 2+ activated enzyme. Total activities increased with increasing concentrations of RuBP to 400 μ m, the assay concentration. However, concentrations above the K m , 25 μ m RuBP, were inhibitory for the initial Rubisco form. Inhibition increased with increasing RuBP concentration. The addition of Mg 2+ in the extraction solution did not prevent RuBP inhibition. The results suggest that the low initial Rubisco activities are principally due to decarbamylation of the active sites of the enzyme during extraction.

Response of Soybean Canopy Photosynthesis to CO2Concentration, Light, and Temperature

Photosynthetic rates of outdoor-grown soybean (Glycine max L. Merr. cv. Bragg) canopies increased with increasing CO2 concentration during growth, before and after canopy closure (complete light interception), when measured over a wide range of solar irradiance values. Total canopy leaf area was greater as the CO2 concentration during growth was increased from 160 to 990 mm3 dm−3. Photosynthetic rates of canopies grown at 330 and 660 mm3 CO2 dm−3 were similar when measured at the same CO2 concentrations and high irradiance. There was no difference in ribulose bisphosphate carboxylase/oxygenase (rubisco) activity or ribulose 1,5-bisphosphate (RuBP) concentration between plants grown at the two CO2 concentrations. However, photosynthetic rates averaged 87% greater for the canopies grown and measured at 660 mm3 CO2 dm−3. A 10°C difference in air temperature during growth resulted in only a 4°C leaf temperature difference, which was insufficient to change the photosynthetic rate or rubisco activity in canopies grown and measured at either 330 or 660 mm3 CO2 dm−3. RuBP concentrations decreased as air temperature during growth was increased at both CO2 concentrations. These data indicate that the increased photosynthetic rates of soybean canopies at elevated CO2 are due to several factors, including: more rapid development of the leaf area index; a reduction in substrate CO2 limitation; and no downward acclimation in photosynthetic capacity, as occur in some other species.

Evidence that Ribulose 1,5-Bisphosphate (RuBP) Binds to Inactive Sites of RuBP Carboxylase in Vivo and an Estimate of the Rate Constant for Dissociation

The binding of ribulose 1,5-bisphosphate (RuBP) to inactive (noncarbamylated) sites of the enzyme RuBP carboxylase in vivo was investigated in Spinacia oleracea and Helianthus annuus. The concentrations of RuBP and inactive sites were determined in leaf tissue as a function of time after a change to darkness. RuBP concentrations fell rapidly after the change to darkness and were approximately equal to the concentration of inactive sites after 60 s. Variations in the concentration of inactive sites, which were induced by differences in the light intensity before the light-dark transition, correlated with the concentration of RuBP between 60 and 120 s after the change to darkness. These data are discussed as evidence that RuBP binds to inactive sites of RuBP carboxylase in vivo. After the concentration of RuBP fell below that of inactive sites (at times longer than 60 s of darkness), the decline in RuBP was logarithmic with time. This would be expected if the dissociation of RuBP from inactive sites controlled the decline in RuBP concentration. These data were used to estimate the rate constant for dissociation of RuBP from inactive sites in vivo.

Evidence for Effects on the in Vivo Activity of Ribulose-Bisphosphate Carboxylase/Oxygenase during Development of Mn Toxicity in Tobacco

The progressive decrease in net photosynthesis accompanying development of Mn toxicity in young leaves of burley tobacco (Nicotiana tabacum L. cv KY 14) is a result of effects on in vivo activity of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (rubisco, EC 4.1.1.39). This conclusion is supported by: (a) decrease in rates of CO(2) depletion during measurements of CO(2) compensation, (b) increase in leaf RuBP concentrations, (c) progressive decreases in rate-constants of RuBP loss (light to dark transition analyses) with progressive increases of leaf Mn concentrations, and (d) restoration of diminished rates of net photosynthesis to control rates by elevated CO(2) (5%). Moreover, elevated CO(2) (1100 microliters per liter) during culture of Mn-treated plants decreased elevated RuBP concentrations to control levels and alleviated foliar symptoms of Mn toxicity. These effects of Mn toxicity on in vivo activity of rubisco were not expressed by in vitro kinetic analyses of rubisco prepared under conditions to sequester Mn or to adsorb polyphenols or their oxidation products. Similarly, the in vitro activity of fructose bisphosphatase (EC 3.1.3.11) was unaffected by Mn toxicity.

Changes in Levels of Intermediates of the C4 Cycle and Reductive Pentose Phosphate Pathway under Various Light Intensities in Maize Leaves

The rate of CO(2) assimilation and levels of metabolites of the C(4) cycle and reductive pentose phosphate pathway in attached leaves of maize (Zea mays L.) were measured over a range of light intensity from 0 to 1,900 microEinsteins per square meter per second under a saturated CO(2) concentration of 350 microliters per liter and a limiting CO(2) concentration of 133 microliters per liter. The level of ribulose 1,5-bisphosphate (RuBP) stayed almost constant (around 60 nanomoles per milligram chlorophyll [Chl]) from low to high light intensities under 350 microliters per liter. Levels of 3-phosphoglycerate (PGA) increased from 100 to 650 nanomoles per milligram Chl under 350 microliters per liter CO(2) with increasing light intensity. The calculated RuBP concentration of 6 millimolar (corresponded to 60 nanomoles per milligram Chl) was about two times above the estimated RuBP binding-site concentration on ribulose bisphosphate carboxylase-oxygenase (Rubisco) of approximately 2.6 millimolar in maize bundle sheath chloroplasts in the light. The ratio of RuBP/PGA increased with decreasing light intensity under 350 microliters per liter CO(2). These results suggest that RuBP carboxylation is under control of light intensity possibly due to a limited supply of CO(2) to Rubisco through the C(4) cycle whose activity is highly dependent on light intensity. Pyruvate level increased with increasing light intensity as long as photosynthesis rate increased. A positive relationship between levels of PGA and those of pyruvate during steady-state photosynthesis under various conditions suggests that an elevated concentration of PGA increases the carbon input into the C(4) cycle through the conversion of PGA to PEP and consequently the level of total intermediates of the C(4) cycle can be raised to mediate higher photosynthesis rate.

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity in Response to Changing Partial Pressure of O2 and Light in Phaseolus vulgaris

The regulation of ribulose-1,5-bisphosphate (RuBP) carboxylase (rubisco) activity in Phaseolus vulgaris was studied under moderate CO(2) and high light, conditions in which photosynthesis in C(3) plants can be insensitive to changes in O(2) partial pressure. Steady state RuBP concentrations were higher, the calculated rate of RuBP use was lower and the activation state of rubisco was lower in low O(2) relative to values observed in normal O(2). It is suggested that the reduced activity of rubisco observed here is related to feedback effects which occur when the rate of net CO(2) assimilation approaches the maximum capacity for starch and sucrose synthesis (triose phosphate utilization). The activation state of rubisco was independent of O(2) partial pressure when light or CO(2) was limiting for photosynthesis. Reduced activity of rubisco was also observed at limiting light. However, in this species light dependent changes in the concentration of an inhibitor of rubisco controlled the apparent V(max) of rubisco in low light while changes in the CO(2)-Mg(2+) dependent activation of rubisco controlled the apparent V(max) in high light.

Photosynthesis and Ribulose 1,5-Bisphosphate Concentrations in Intact Leaves of Xanthium strumarium L.

The interacting effects of the rate of ribulose 1,5-bisphosphate (RuBP) regeneration and the rate of RuBP utilization as influenced by the amount and activation of RuBP carboxylase on photosynthesis and RuBP concentrations were resolved in experiments which examined the kinetics of the response of photosynthesis and RuBP concentrations after step changes from a rate-saturating to a rate-limiting light intensity in Xanthium strumarium. Because RuBP carboxylase requires several minutes to deactivate in vivo, it was possible to observe the effect of reducing the rate of RuBP regeneration on the RuBP concentration at constant enzyme activation state by sampling very soon after reducing the light intensity. Samples taken over longer time periods showed the effect of changes in enzyme activation at constant RuBP regeneration rate on RuBP concentration and photosynthetic rate. Within 15 s of lowering the light intensity from 1500 to 600 microEinsteins per square meter per second the RuBP concentration in the leaves dropped below the enzyme active site concentration, indicating that RuBP regeneration rate was limiting for photosynthesis. After longer intervals of time, the RuBP concentration in the leaf increased as the RuBP carboxylase assumed a new steady state activation level. No change in the rate of photosynthesis was observed during the interval that RuBP concentration increased. It is concluded that the rate of photosynthesis at the lower light intensity was limited by the rate of RuBP regeneration and that parallel changes in the activation of RuBP carboxylase occurred such that concentrations of RuBP at steady state were not altered by changes in light intensity.

Species variation in kinetic properties of ribulose 1,5-bisphosphate carboxylase/oxygenase

Several kinetic parameters of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase from different species were measured and compared. The CO 2 O 2 specificity ( V cK o V oK c ) was found to be about 80 in the enzymes from several C 3 species and two C 4 species. Specificity values of 58 and 70, respectively, were found in enzymes from the C 4 plants Setaria italica and Sorghum bicolor. Two enzymes from cyanobacteria had values of about 50. Substitution of Mn 2+ for Mg 2+ reduced the CO 2 O 2 specificity by a factor of about 20 for all enzymes except that of Rhodospirillum rubrum, which was reduced by a factor of 10. Values for K Mg 2+(apparent) measured at 102 μ m CO 2 were found to vary by a factor of 8 between different RuBP carboxylase/oxygenase enzymes. Enzymes with high K Mg 2+(apparent) values generally had high Michaelis constants for CO 2. The rate of CO 2 Mg 2+ activation was inhibited by RuBP in all enzymes, although the concentration of RuBP required to inhibit activation in the enzyme from the cyanobacterium Aphanizomenon flos-aquae was increased by an order of magnitude compared to other higher plant structural-type enzymes. The wide variation found in the kinetic properties of RuBP carboxylase/oxygenase isolated from diverse species appears to be determined in part by past evolutionary pressures and the present physiocochemical environment in which the enzyme functions.

Oxygen and Carbon Dioxide Effects on the Pool Size of Some Photosynthetic and Photorespiratory Intermediates in Soybean (Glycine max [L.] Merr.)

The levels of ribulose 1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (PGA), glycolate, glycine, and serine were measured in soybean leaflets during photosynthesis in atmospheres ranging from 1 to 60% O(2) and from 0 to 500 microliters per liter CO(2.)The RuBP level remained constant as CO(2) concentration was decreased in atmospheres containing 20 or 60% O(2), but increased as CO(2) concentration was decreased in atmospheres containing 1% O(2.) PGA levels decreased at CO(2) concentrations near or below the CO(2) compensation point under all O(2) concentrations. The glycolate pool at 300 microliters per liter CO(2) increased slightly with increasing O(2) concentration, but remained nearly constant at very low CO(2). The serine pool showed no measurable change over the range of CO(2) or O(2) concentrations tested. The glycine pool did not change significantly with varying CO(2) concentration but increased linearly with increasing O(2) concentration.Measured RuBP levels indicate an RuBP concentration less than the estimated concentration of RuBP carboxylase/oxygenase active sites. The constant RuBP pool size in 20% O(2), however, indicates that RuBP level does not limit photosynthesis or photorespiration any more at 50 microliters per liter CO(2) than at 450 microliters per liter.