Ribulose 1,5-bisphosphate carboxylase/oxygenase from Pseudomonas oxalacticus.

Ribulose 1,5-bisphosphate carboxylase/oxygenase was purified by a rapid, facile procedure from formate-grown Pseudomonas oxalaticus. The electrophoretically homogeneous enzyme had specific activities of 1.9 mumol of CO2 fixed per min per mg of protein and 0.15 mumol of O2 consumed per min per mg of protein. The amino acid composition was similar to that of other bacterial sources of the enzyme. The molecular weights determined by sedimentation equilibrium and by gel filtration were 421,000 and 450,000, respectively. Upon sodium dodecyl sulfate electrophoresis of enzyme purified under conditions which would limit proteolysis, two types of large (L) subunits and two types of small (S) subunits were observed with apparent molecular weights of 57,000, 55,000, 17,000 and 15,000. By densitometric scans at two different protein concentrations the stoichiometry of the total large to total small subunits was 1:1, implying an L6S6 structure. Electron micrographs of the enzyme revealed an unusual structure that was inconsistent with a cubical structure. The enzyme had an unusually high Km for ribulose 1,5-bisphosphate (220 microM) and was strongly inhibited by 6-phosphogluconate in the ribulose 1,5-bisphosphate carboxylase assay (Ki = 270 microM). One, 5, and 12 days after purification the enzyme was half-maximally activated at 0.13 microM, 0.23 mM, and 0.70 mM CO2, respectively, at saturating Mg2+. At saturating CO2, enzyme 1 day afer purification responded sigmoidally to Mg2+ and was half-maximally activated by 0.85 mM Mg2+ in the absence of 6-phosphogluconate (Hill coefficient, h = 2.0) and by 0.19 mM Mg2+ in the presence of mM 6-phosphogluconate (h = 1.7).     

Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by ribulose-1,5-bisphosphate epimerization and degradation products

Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P₂) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P₂, is also present. Both compounds simulate the substrate inhibition of ribulose-P₂ carboxylase/oxygenase previously reported for ribulose-P₂. Freshly prepared ribulose-P₂ had little inhibitory activity. The instability of ribulose-P₂ may be one reason for a high level of ribulose-P₂ carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P₂. Because the K_D of the enzyme/substrate complex is ≤1 μM, all ribulose-P₂ generated in situ may be stored as this complex to prevent decomposition.     

Inhibition and stimulation of ribulose-1,5-bisphosphate carboxylase/oxygenase by glyoxylate

Glyoxylate is a slowly reversible inhibitor of the CO2/Mg2+-activated form of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach leaves. Inactivation occurred with an apparent dissociation constant of 3.3 mM and a maximum pseudo-first-order rate constant of 7 X 10(-3) s-1. The rate constant for reactivation was 1.2 X 10(-2) s-1. Glyoxylate did not cause differential inhibition of ribulosebisphosphate carboxylase or oxygenase activities. 6-Phosphogluconate protected the enzyme from inactivation by glyoxylate. Glyoxylate was incorporated irreversibly into the large subunit of ribulosebisphosphate carboxylase after reduction with sodium borohydride. Activated enzyme incorporated 1.3 mol of glyoxylate per mole protomer, while enzyme treated with carboxyarabinitol 1,5-bisphosphate (CABP) to protect the active sites incorporated only 0.3 mol glyoxylate per mole protomer. The data suggest that glyoxylate forms a Schiff base with a lysyl residue in the region of the catalytic site. Glyoxylate stimulated the activity of the unactivated enzyme by about twofold. Pseudo-first-order inactivation also occurred with the unactivated enzyme after the initial stimulation by glyoxylate, although at a much slower rate than with the activated enzyme. Glyoxylate treatment of partially activated enzyme did not stimulate formation of the quaternary complex of enzyme X CO2 X Mg2+ X CABP.     

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.     

Inhibition of ribulose 1,5-bisphosphate carboxylase/oxygenase by 2-carboxyarabinitol-1-phosphate

In some plants, 2-carboxy-d-arabinitol 1-phosphate (CA 1P) is tightly bound to catalytic sites of ribulose, 1,5-bisphosphate carboxylase/oxygenase (rubisco). This inhibitor's tight binding property results from its close resemblance to the transition state intermediate of the carboxylase reaction. Amounts of CA 1P present in leaves varies with light level, giving CA 1P characteristics of a diurnal modulator of rubisco activity. Recently, a specific phosphatase was found that degrades CA 1P, providing a mechanism to account for its disappearance in the light. The route of synthesis of CA 1P is not known, but could involve the branched chain sugar, hamamelose. There appear to be two means for diurnal regulation of the number of catalytic sites on rubisco: carbamylation mediated by the enzyme, rubisco activase, and binding of CA 1P. While strong evidence exists for the involvement of rubisco activase in rubisco regulation, the significance of CA 1P in rubisco regulation is enigmatic, given the lack of general occurrence of CA 1P in plant species. Alternatively, CA 1P may have a role in preventing the binding of metabolites to rubisco during the night and the noncatalytic binding of ribulose bisphosphate in the light.     

Ribulose 1,5-bisphosphate carboxylase and phosphoribulokinase in Prochloron

Cell-free extracts of Prochloron didemni were assayed for ribulose 1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) and phosphoribulokinase (EC 2.7.1.19), two key enzymes in the reductive pentose-phosphate cycle. In an RuBP-dependent reaction, the production of two molecules of 3-phosphoglycerate per molecule of CO₂ fixed was shown. Phosphoribulokinase activity was demonstrated by the production of ADP from ribulose 5-phosphate (Ru5P) and ATP and by measurement of ATP-, Ru5P-dependent ¹⁴CO₂ fixation in the presence of excess spinach RuBP carboxylase. When Prochloron RuBP carboxylase was purified from cell-free extracts by isopycnic centrifugation in reoriented linear 0.2 to 0.8 M sucrose gradients, the enzyme sedimented to a position which corresponded to that for the 520,000-dalton spinach enzyme. After polyacrylamide gel electrophoresis (PAGE) of Prochloron enzyme, a major band of enzyme activity corresponded to that for the spinach enzyme. Considerably more additional carboxylase activity was found in a less mobile species than was the case for spinach RuBP carboxylase. Sodium dodecyl sulfate-PAGE of the Prochloron enzyme indicates that it is composed of both large (molecular weight, MW=57,500) and small (MW=18,800) subunits.     

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.     

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/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.     

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.     

Ribulose 1,5-bisphosphate and activation of the carboxylase in the chloroplast

Ribulose 1,5-bisphosphate in the chloroplast has been suggested to regulate the activity of the ribulose bisphosphate carboxylase/oxygenase. To generate high levels of ribulose bisphosphate, isolated and intact spinach chloroplasts were illuminated in the absence of CO₂. Under these conditions, chloroplasts generate internally up to 300 nanomoles ribulose 1,5-bisphosphate per milligram chlorophyll if O₂ is also absent. This is equivalent to 12 millimolar ribulose bisphosphate, while the enzyme, ribulose bisphosphate carboxylase, offers up to 3.0 millimolar binding sites for the bisphosphate in the chloroplast stroma. During illumination, the ribulose bisphosphate carboxylase is deactivated, due mostly to the absence of CO₂ required for activation. The rate of deactivation of the ribulose bisphosphate carboxylase was not affected by the chloroplast ribulose bisphosphate levels. Upon addition of CO₂, the carboxylase in the chloroplast was completely reactivated. Of interest, addition of 3-phosphoglycerate stopped deactivation of the carboxylase in the chloroplast while ribulose bisphosphate accumulated. With intact chloroplasts in light, no correlation between deactivation of the carboxylase and ribulose bisphosphate levels could be shown.     

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.     

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.     

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.