Ribulose 1,5-bisphosphate carboxylase and oxygenase from Thiocapsa roseopersicina: activation and catalysis.

Ribulose 1,5-bisphosphate carboxylase/oxygenase purified from malate-grown Thiocapsa roseopersicina required Mg²⁺ for the activation of both carboxylase and oxygenase activities. Mg²⁺ was either not required or required at very low concentrations for catalysis by both enzyme activities. EDTA and dithiothreitol had no effect on ribulose 1,5-biphosphate oxygenase. The K_0.5 values with respect to Mg²⁺ for activation of the carboxylase and oxygenase activities were 8.4 and 2 mm, respectively. Ribulose 1,5-biphosphate carboxylase and oxygenase activities revealed differential sensitivities to 6-phosphogluconate. This ligand at 1 mm inhibited the carboxylase activity 30%, whereas the oxygenase activity was inhibited by 69%.     

Dissociation of ribulose-1,5-bisphosphate bound to ribulose-1,5-bisphosphate carboxylase/oxygenase and its enhancement by ribulose-1,5-bisphosphate carboxylase/oxygenase activase-mediated hydrolysis of ATP

Ribulose bisphosphate (RuBP), a substrate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is an inhibitor of Rubisco activation by carbamylation if bound to the inactive, noncarbamylated form of the enzyme. The effect of Rubisco activase on the dissociation kinetics of RuBP bound to this form of the enzyme was examined and characterized with the use of ³H-labeled RuBP and proteins purified from spinach (Spinacia oleracea L.) In the absence of Rubisco activase and in the presence of a large excess of unlabeled RuBP, the dissociation rate of bound [1-³H]RuBP was much faster after a short (30 second) incubation than after an extended incubation (1 hour). After 1 hour of incubation, the dissociation rate constant (K_off) of the bound RuBP was 4.8 × 10⁻⁴ per second, equal to a half-time of about 35 minutes, whereas the rate after only 30 seconds was too fast to be accurately measured. This time-dependent change in the dissociation rate was reflected in the subsequent activation kinetics of Rubisco in the presence of RuBP, CO₂, and Mg²⁺, and in both the absence or presence of Rubisco activase. However, the activation of Rubisco also proceeded relatively rapidly without Rubisco activase if the RuBP level decreased below the estimated catalytic site concentration. High pH (pH 8.5) and the presence of Mg²⁺ in the medium also enhanced the dissociation of the bound RuBP from Rubisco in the presence of RuBP. In the presence of Rubisco activase, Mg²⁺, ATP (but not the nonhydrolyzable analog, adenosine-5′-O-[3-thiotriphosphate]), excess RuBP, and an ATP-regenerating system, the dissociation of [1-³H]RuBP from Rubisco was increased in proportion to the amount of Rubisco activase added. This result indicates that Rubisco activase-mediated hydrolysis of ATP is required for promotion of the enhanced dissociation of the bound RuBP from Rubisco. Furthermore, product analysis by ion-exchange chromatography demonstrated that the release of the bound RuBP, in an unchanged form, was considerably faster than the observed increase in Rubisco activity. Thus, RuBP dissociation was experimentally separated from activation and precedes the subsequent formation of active, carbamylated Rubisco during activation of Rubisco by Rubisco activase.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase protein prevents the in vitro decline in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase

The rate of CO₂ fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) following addition of ribulose 1,5-bisphosphate (RuBP) to fully activated enzyme, declined with first-order kinetics, resulting in 50% loss of rubisco activity after 10 to 12 minutes. This in vitro decline in rubisco activity, termed fall-over, was prevented if purified rubisco activase protein and ATP were added, allowing linear rates of CO₂ fixation for up to 20 minutes. Rubisco activase could also stimulate rubisco activity if added after fallover had occurred. Gel filtration of the RuBP-rubisco complex to remove unbound RuBP allowed full activation of the enzyme, but the inhibition of activated rubisco during fallover was only partially reversed by gel filtration. Addition of alkaline phosphatase completely restored rubisco activity following fallover. The results suggest that fallover is not caused by binding of RuBP to decarbamylated enzyme, but results from binding of a phosphorylated inhibitor to the active site of rubisco. The inhibitor may be a contaminant in preparations of RuBP or may be formed on the active site but is apparently removed from the enzyme in the presence of the rubisco activase protein.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from parsley.

Ribulose-1,5-bisphosphate carboxylase/oxygenase from parsley leaves was purified by Sepharose 6B gel filtration at pH 8.3 as a single, colorless peak containing both activities. Approximately 0.2 g atom copper per mole enzyme was detected by atomic absorption spectroscopy, but this copper was not detectable by EPR spectrometry.     

Aldolase--An Important Enzyme in Controlling the Ribulose 1,5-bisphosphate Regeneration Rate in Photosynthesis

This study was done to explore an enzymatic mechanism for thephotosynthetic carbon reduction cycle whereby the rate of synthesisof ribulose 1,5-bisphosphate (RuBP) could be changed while thelevels of intermediates other than 3-phosphoglycerate and RuBPwere kept constant. Chloroplast aldolase was purified to homogeneityfrom spinach leaves. When the enzyme was assayed in the directionof fructose 1,6-bisphosphate synthesis in the presence of theconcentrations of the substrates reported in vivo, the activitywas severely inhibited by physiological concentrations of RuBP.The aldolase reaction proceeded with a sequential mechanism.The K_m for dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphatewere 0.45 mM and 40 µM, respectively. The activity wascompetitively inhibited by RuBP with respect to dihydroxyacetonephosphate. The K^I was 0.78 mM. The maximum activity of aldolasein spinach leaves was calculated as 1,360µmol (mg Chl)^–1h^–1 An equation to express the reaction for the synthesisof fructose 1,6-bisphosphate by aldolase was constructed topredict the metabolic rate of this reaction in vivo. The calculationclearly showed that aldolase is an important enzyme in controllingthe rate of RuBP regeneration. (Received March 25, 1991; Accepted August 12, 1991)     

Ribulose 1,5-bisphosphate carboxylase and polyhedral bodies of Chlorogloeopsis fritschii

Ribulose 1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) activity was approximately equally distributed between supernatant and pellet fractions produced by differential centrifugation of disrupted cells of Chlorogloeopsis fritschii. Low ionic strength buffer favoured the recovery of particulate RuBP carboxylase. Density gradient centrifugation of resuspended cell-free particulate material produced a single band of RuBP carboxylase activity, which was associated with the polyhedral body fraction, rather than with the thylakoids or other observable particles. Isolated polyhedral body stability was improved by density gradient centrifugation through gradients of Percoll plus sucrose in buffer, which yielded apparently intact polyhedral bodies. These were 100 to 150 nm in diameter and contained ring-shaped, 12 nm diameter particles. It is inferred that the C. fritschii polyhedral bodies are carboxysomes. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of SDS-dissociated polyhedral bodies revealed 8 major polypeptides. The most abundant, with molecular weights of 52,000 and 13,000, correspond with the large and small subunits, respectively, of RuBP carboxylase.Abbreviations RuBP ribulose 1,5-bisphosphate - Ru5P ribulose 5-phosphate - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediamine tetraacetic acid - Tris tris (hydroxymethyl) methylamine - IB isolation buffer - TCA trichloroacetic acid     

Limiting Factors in Photosynthesis: VI. Regeneration of Ribulose 1,5-Bisphosphate Limits Photosynthesis at Low Photochemical Capacity

Earlier work (SE Taylor, N Terry [1984] Plant Physiol 75: 82-86) has shown that the rate of photosynthesis may be colimited by photosynthetic electron transport capacity, even at low intercellular CO₂ concentrations. Here we monitored leaf metabolites diurnally and the activities of key Calvin cycle enzymes in the leaves of three treatment groups of sugar beet (Beta vulgaris L.) plants representing three different in vivo photochemical capacities, i.e. Fe-sufficient (control) plants, moderately Fe-deficient, and severely Fe-deficient plants. The results show that the decrease in photosynthesis with Fe deficiency mediated reduction in photochemical capacity was through a reduction in ribulose 1,5-bisphosphate (RuBP) regeneration and not through a decrease in ribulose 1,5-bisphosphate carboxylase/oxygenase activity. Based on measurements of ATP and NADPH and triose phosphate/3-phosphoglycerate ratios in leaves, there was little evidence that photosynthesis and RuBP regeneration in Fe-deficient leaves were limited directly by the supply of ATP and NADPH. It appeared more likely that photochemical capacity influenced RuBP regeneration through modulation of enzymes in the photosynthetic carbon reduction cycle between fructose-6-phosphate and RuBP; in particular, the initial activity of ribulose-5-phosphate kinase was strongly diminished by Fe deficiency. Starch and sucrose levels changed independently of one another to some extent during the diurnal period (both increasing in the day and decreasing at night) but the average rates of starch or sucrose accumulation over the light period were each proportional to photochemical capacity and photosynthetic rate.     

Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities

An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-¹⁴C,5-³H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate V_c/V_o ratios from the expression A/(B-A) where A and B represent the ³H/¹⁴C isotope ratios of doubly labeled RuBP and 3-PGA, and V_c and V_o represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-¹⁴C]glucose and [6-³H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase.     

Activity of ribulose 1,5-bisphosphate carboxylase oxygenase as a function of storage conditions

Optimal storage conditions to retain ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity were investigated. The soluble spinach (Spinacia oleracea) enzyme was pretreated with its activators, Mg²⁺ and HCO₃⁻, and then stored for up to 30 days at 4 or −18°C or in liquid N₂. Cold inactivation and conformational changes were suggested to be involved during Rubisco storage in the cold, leading to its inactivation. Pretreatment of the enzyme with Mg²⁺ and CO₂ and subsequent storage at either 4°C or in liquid N₂ or flushing the samples with N₂ and rapid freezing and storage in liquid N₂ are recommended as storage procedures. These storage treatments will prevent inactivation, so that full original specific activity will be preserved.     

Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: A different perspective

Marine and terrestrial photosynthetic and chemoautotrophic microorganisms assimilate considerable amounts of carbon dioxide. Like green plastids, the predominant means by which this process occurs is via the Calvin-Benson-Bassham reductive pentose phosphate pathway, where ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a paramount role. Recent findings indicate that this enzyme is subject to diverse means of control, including specific and elaborate means to guarantee its high rate and extent of synthesis. In addition, powerful and specific means to regulate Rubisco activity is a characteristic feature of many microbial systems. In many respects, the diverse properties of microbial Rubisco enzymes suggest interesting strategies to elucidate the molecular basis of CO₂/O₂ specificity, the holy grail of Rubisco biochemistry. These systems thus provide, as the title suggests, different perspectives to this fundamental problem. These include vast possibilities for imaginative biological selection using metabolically versatile organisms with well-defined genetic transfer capabilities to solve important issues of Rubisco specificity and molecular control. This review considers the major issues of Rubisco biochemistry and regulation in photosynthetic microoganisms including proteobacteria, cyanobacteria, marine nongreen algae, as well as other interesting prokaryotic and eukaryotic microbial systems recently shown to possess this enzyme.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from thermophilic cyanobacterium Thermosynechococcus elongatus

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) can be divided into two branches: the “red-like type” of marine algae and the “green-like type” of cyanobacteria, green algae, and higher plants. We found that the “green-like type” rubisco from the thermophilic cyanobacterium Thermosynechococcus elongatus has an almost 2-fold higher specificity factor compared with rubiscos of mesophilic cyanobacteria, reaching the values of higher plants, and simultaneously revealing an improvement in enzyme thermostability. The difference in the activation energies at the transition stages between the oxygenase and carboxylase reactions for Thermosynechococcus elongatus rubisco is very close to that of Galdieria partita and significantly higher than that of spinach. This is the first characterization of a “green-like type” rubisco from thermophilic organism.     

Loose association of ribulose 1,5-bisphosphate carboxylase/oxygenase with chloroplast thylakoid membranes

The intra-chloroplastic distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) between thylakoid membranes and stroma was studied by determining the enzyme activities in the two fractions, obtained by the rapid centrifugation of hypotonically disrupted chloroplast preparations of spinach and pea leaf tissues. The membrane-associated form of RuBisCO was found to increase in proportion to the concentration of MgCl₂ in the disrupting medium; with 20 mM MgCl₂ approximately 20% of the total RuBisCO of spinach chloroplasts and 10% of that of pea chloroplasts became associated with thylakoid membranes. Once released from membranes in the absence of MgCl₂, addition of MgCl₂ did not cause reassociation of the enzyme. The inclusion of KCl in the hypotonic disruption buffer also caused the association of RuBisCO with membranes; however, up to 30 mM KCl, only minimal enzyme activities could be detected in the membranes, whereas above 40 mM KCl there was a sharp increase in the membrane-associated form of the enzyme.Higher concentrations of chloroplasts during the hypotonic disruption, as well as addition of purified preparations of RuBisCO to the hypotonic buffer, resulted in an increase of membrane-associated activity. Therefore, the association of the enzyme with thylakoid membranes appears to be dependent on the concentration of RuBisCO. P-glycerate kinase and aldolase also associated to the thylakoid membranes but NADP-linked glyceraldehyde-3-P dehydrogenase did not. The optimal conditions for enzyme association with the thylakoid membranes were examined; maximal association occurred at pH 8.0. The association was temperature-insensitive in the range of 4° to 25° C. RuBisCO associated with the thylakoid membranes could be gradually liberated to the soluble form upon shaking in a Vortex mixer at maximal speed, indicating that the association is loose.Abbreviations DTT dithiothreitol - RuBP ribulose 1,5-bisphosphate - RuBisCO ribulose 1,5-bisphosphate carboxylase/oxygenase - MES 2-(N-morpholino) ethane sulfonic acid     

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase deficiency delays senescence of ribulose-1,5-bisphosphate carboxylase/oxygenase but progressively impairs its catalysis during tobacco leaf development.

Transgenic tobacco (Nicotiana tabacum L. cv W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) activase grew more slowly than wild-type plants in a CO2-enriched atmosphere, but eventually attained the same height and number of leaves. Compared with the wild type, the anti-activase plants had reduced CO2 assimilation rates, normal contents of chlorophyll and soluble leaf protein, and much higher Rubisco contents, particularly in older leaves. Activase deficiency greatly delayed the usual developmental decline in Rubisco content seen in wild-type leaves. This effect was much less obvious in another transgenic tobacco with an antisense gene directed against chloroplast-located glyceraldehyde-3-phosphate dehydrogenase, which also had reduced photosynthetic rates and delayed development. Although Rubisco carbamylation was reduced in the anti-activase plants, the reduction was not sufficient to explain the reduced photosynthetic rate of older anti-activase leaves. Instead, up to a 10-fold reduction in the catalytic turnover rate of carbamylated Rubisco in vivo appeared to be the main cause. Slower catalytic turnover by carbamylated Rubisco was particularly obvious in high-CO2-grown leaves but was also detectable in air-grown leaves. Rubisco activity measured immediately after rapid extraction of anti-activase leaves was not much less than that predicted from its degree of carbamylation, ruling out slow release of an inhibitor from carbamylated sites as a major cause of the phenomenon. Nor could substrate scarcity or product inhibition account for the impairment. We conclude that activase must have a role in vivo, direct or indirect, in promoting the activity of carbamylated Rubisco in addition to its role in promoting carbamylation.     

Modification of carboxyl groups at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach was inactivated by a carboxyl-directed reagent, Woodward's reagent K ( WRK ). The inactivation followed pseudo-first-order kinetics. The reaction order with respect to inactivation by WRK was 1.1, suggesting that inactivation was the consequence of modifying a single residue per active site. The substrate ribulose 1,5-bisphosphate (RBP), two competitive inhibitors, fructose 1,6-bisphosphate (FBP) and sedoheptulose 1,7-bisphosphate (SBP), and a number of sugars-phosphate protected against inactivation by WRK . SBP was a strong protector, displaying a dissociation constant (Kd) of 3 microM with native RBP carboxylase. Pretreatment of RBP carboxylase with diethyl pyrocarbonate prevented WRK incorporation into the enzyme. The enol ester derivative produced by reaction of WRK with RBP carboxylase has a maximal absorbance at 346 nm, and the extinction coefficient was found to be 12300 +/- 700 M-1 cm-1. Spectrophotometric titration of the number of carboxyl groups modified by WRK in RBP carboxylase/oxygenase in the presence and in the absence of SBP suggests that inactivation was associated with the modification of one carboxyl group per active site.     

Dissociation of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach by urea

The dissociation of D-ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach, which consists of eight large subunits (L, 53 kDa) and eight small subunits (S, 14 kDa) and thus has a quarternary structure L8S8, has been investigated using a variety of physical techniques. Gel chromatography using Sephadex G-100 indicates the quantitative dissociation of the small subunit S from the complex at 3-4 M urea (50 mM Tris/Cl pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol and 5 mM 2-mercaptoethanol). The dissociated S is monomeric. Analytical ultracentrifuge studies show that the core of large subunits, L, remaining at 3-4 M urea sediments with S20, w = 15.0 S, whereas the intact enzyme (L8S8) sediments with S20, w = 17.7S. The observed value is consistent with a quarternary structure L8. The dissociation reaction in 3-4 M urea can thus be represented by L8S8----L8 + 8S. At urea concentrations c greater than 5 M the L8 core dissociates into monomeric, unfolded large subunits. A large decrease in fluorescence emission intensity accompanies the dissociation of the small subunit S. This change is completed at 4 M urea. No changes are observed upon dissociating the L8 core. The kinetics of dissociation of the small subunit, as monitored by fluorescence spectroscopy, closely follow the kinetics of loss of carboxylase activity of the enzyme. Studies of the circular dichroism of D-ribulose-1,5-bisphosphate carboxylase in the wavelength region 200-260 nm indicate two conformational transitions. The first one ([0]220 from -8000 to -3500 deg cm2 dmol-1) is completed at 4 M urea and corresponds to the dissociation of the small subunit and coupled conformational changes. The second one ([0]220 from -3500 to -1200 deg cm2 dmol-1) is completed at 6 M urea and reflects the dissociation and unfolding of large subunits from the core. The effect of activation of the enzyme by addition of MgCl2 (10 mM) and NaHCO3 (10 mM) on these conformational transitions was investigated. The first conformational transition is then shifted to higher urea concentrations: a single transition ([0]220 from -8000 to -1200 deg cm2 dmol-1) is observed for the activated enzyme. From the urea dissociation experiments we conclude that both large (L) and small (S) subunits are important for carboxylase activity of spinach D-ribulose-1,5-bisphosphate carboxylase: the L-S subunit interactions tighten upon activation and dissociation of S leads to a coupled, proportional loss of enzyme activity.     

Activation-dependent changes in microenvironment of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase

The spinach ribulose 1,5-bisphosphate carboxylase/oxygenase was labelled with o-phthalaldehyde, which forms a stable fluorescent isoindole adduct at the active site. The fluorescence behaviour of the labelled enzyme after activation to different levels by Mg2+ was compared with that of a synthetic isoindole adduct of o-phthalaldehyde, namely 1-(hydroxyethylthio)-2-beta hydroxyethylisoindole in solvents of different pH and polarity. The results suggest that the microenvironment at the catalytically incompetent active site of the unactivated Rubisco is highly acidic (pH less than 2) in nature. The activation by Mg2+ results in the conformational change such that the effective pH at the active site increases to greater than 8. The polarity of the active site of the activated enzyme was found to be similar to that of a mixture of hexane and toluene.     

Questions about the complexity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) has played a central role in our understanding of chloroplast biogenesis and photosynthesis. In particular, its catalysis of the rate-limiting step of CO₂ fixation, and the mutual competition of CO₂ and O₂ at the active site, makes Rubisco a prime focus for genetically engineering an increase in photosynthetic productivity. Although it remains difficult to manipulate the chloroplast-encoded large subunit and nuclear-encoded small subunit of crop plants, much has been learned about the structure/function relationships of Rubisco by expressing prokaryotic genes in Escherichia coli or by exploiting classical genetics and chloroplast transformation of the green alga Chlamydomonas reinhardtii. However, the complexity of chloroplast Rubisco in land plants cannot be completely addressed with the existing model organisms. Two subunits encoded in different genetic compartments have coevolved in the formation of the Rubisco holoenzyme, but the function of the small subunit remains largely unknown. The subunits are posttranslationally modified, assembled via a complex process, and degraded in regulated ways. There is also a second chloroplast protein, Rubisco activase, that is responsible for removing inhibitory molecules from the large-subunit active site. Many of these complex interactions and processes display species specificity. This means that attempts to engineer or discover a better Rubisco may be futile if one cannot transfer the better enzyme to a compatible host. We must frame the questions that address this problem of chloroplast-Rubisco complexity. We must work harder to find the answers.