Storage and maintaining activity of ribulose bisphosphate carboxylase/oxygenase

Purified ribulose-1,5-bisphosphate carboxylase/oxygenase in 50% saturated (NH₄)₂SO₄ was stable when frozen as small beads in liquid nitrogen and stored at −80 C. When stored as a slurry at 4 C most of the activity was lost within four weeks. This loss was due not only to enzyme polymerization. Activity in old preparations purified from spinach leaves, but not tobacco or tomato leaves, can be restored to the level of newly purified enzyme after storage at 4 C by treatment with 50 to 100 millimolar dithiothreitol for several hours followed by dialysis against buffer and 1 millimolar dithiothreitol before CO₂ and Mg²⁺ activation and assay. Some enzyme oligomers that had been formed were not converted back to native enzyme by treatment with 100 millimolar dithiothreitol.     

Reduction of ribulose biphosphate carboxylase activase levels in tobacco (Nicotiana tabacum) by antisense RNA reduces ribulose biphosphate carboxylase carbamylation and impairs photosynthesis.

The in vivo activity of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is modulated in response to light intensity by carbamylation of the active site and by the binding of sugar phosphate inhibitors such as 2'-carboxyarabinitol-1-phosphate (CA 1P). These changes are influenced by the regulatory protein Rubisco activase, which facilitates the release of sugar phosphates from Rubisco's catalytic site. Activase levels in Nicotiana tabacum were reduced by transformation with an antisense gene directed against the mRNA for Rubisco activase. Activase-deficient plants were photosynthetically impaired, and their Rubisco carbamylation levels declined upon illumination. Such plants needed high CO2 concentrations to sustain reasonable growth rates, but the level of carbamylation was not increased by high CO2. The antisense plants had, on average, approximately twice as much Rubisco as the control plants. The maximum catalytic turnover rate (k cat) of Rubisco decreases in darkened tobacco leaves because of the binding of CA 1P. The dark-to-light increase in k cat that accompanies CA 1P release occurred to similar extents in antisense and control plants, indicating that normal levels of activase were not essential for CA 1P release from Rubisco in the antisense plants. However, CA 1P was released in the antisense plants at less than one-quarter of the rate that it was released in the control plants, indicating a role for activase in accelerating the release of CA 1P.     

Trapping of a reaction intermediate by tetranitromethane during catalysis by ribulose biphosphate carboxylase

Illumination at 230 K of dithionite-reduced particles results in the appearance of an EPR detectable radical 13 G wide with g = 2.0033. This radical is formed in a ratio of 2.28 (±0.5)/P700. Investigation of the time course of formation shows two components are present. One (A₁) has g = 2.0051 and the other (A_og= 2.0024. Reduction of A₁ results in an increase in reaction centre triplet formation, subsequent reduction of A_o results in a decrease of triplet formation to the base level. We propose that these components function sequentially in the transfer of electrons from P700 to the iron—sulphur acceptors.     

The effects of bivalent cations on ribulose bisphosphate carboxylase/oxygenase.

The half-saturation constants for binding of the bivalent cations (Mg2+, Ni2+, Co2+, Fe2+ and Mn2+) to ribulose bisphosphate carboxylase/oxygenase from Glycine max and Rhodospirillum rubrum were measured. The values obtained were dependent on the enzyme and the cation present, but were the same for both oxygenase and carboxylase activities. Ribulose bisphosphate rather than its cation complex was the true substrate. The kinetic parameters Vmax.(CO2), Vmax.(O2), Km(CO2), Km(O2), and K1(O2) were determined for both enzymes and each cation activator. The evolutionary and mechanistic implications of these data are discussed.     

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.     

Changing ribulose diphosphate carboxylase/oxygenase activity in ripening tomato fruit

Tomato fruit (Lycopersicum esculentum Mill) from green, pink, and red stages were assayed for changes in the activity of ribulose diphosphate carboxylase and oxygenase, phosphoenolpyruvate carboxylase, changes in the levels of glycolate and respiratory gas exchange. The ribulose diphosphate carboxylase activity decreased as the fruit ripened. By comparison, the ribulose diphosphate oxygenase activity increased during the transition from the green to the pink stage, and declined afterward. The changes in the endogenous glycolate levels and the respiratory gas exchange, as observed at different stages of ripening, resembled the changes in the ribulose diphosphate oxygenase activity. The utilization of glycolate in further metabolic activity may result in the formation of peroxidases required for the onset of ripening.     

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.     

The dimerization of folded monomers of ribulose 1,5-bisphosphate carboxylase/oxygenase

Spontaneous refolding and reconstitution processes of dimeric ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum have been investigated using size-exclusion high performance liquid chromatography (HPLC), spectroscopic, and activity measurements. When the unfolded Rubisco in guanidine hydrochloride is diluted at 4 degrees C, a folding intermediate (Rubisco-I) is rapidly formed, which remains in an unstable monomeric state and gradually develops into folded monomer (Rubisco-M) at 4 degrees C but undergoes irreversible aggregation at 25 degrees C. Refolding of Rubisco-I to Rubisco-M is a very slow process, taking about 20 h for 70% conversion at 4 degrees C. Rubisco-M is stable at 4 degrees C and is capable of forming an active dimer spontaneously when incubated at a temperature higher than 10 degrees C. The dynamic dimerization process has been measured in a temperature range of 4-35 degrees C by HPLC, and the results demonstrate that the dimerization is strongly facilitated by the temperature. It is found that dithiothreitol is essential for the spontaneous reconstitution of Rubisco.     

Prospects for manipulating the substrate specificity of ribulose bisphosphate carboxylase/oxygenase

Pierce, J. 1988. Prospects for manipulating the substrate specificity of ribulose bisphosphate carboxylase/oxygenase. - Physiol. Plant. 72: 690–698.
The idea of enhancing plant productivity by minimizing the apparently wasteful process of photorespiration has been an enduring one. Since the relative fluxes of carbon through the competing pathways of photosynthesis and photorespiration are determined by the kinetic properties of a single enzyme, ribulose bisphosphate carboxylase/oxygenase, it has been conjectured that genetic modification of this protein could provide more productive plants. Recent advances in techniques for studying ribulose bisphosphate carboxylase/oxygenase hold promise for determining whether such modifications will prove possible.     

Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants

Transgenic manipulation of the photosynthetic CO2-fixing enzyme, ribulose bisphosphate carboxylase/oxygenase (Rubisco) in higher plants provides a very specific means of testing theories about photosynthesis and its regulation. It also encourages prospects for radically improving the efficiencies with which photosynthesis and plants use the basic resources of light, water, and nutrients. Manipulation was once limited to variation of the leaf's total content of Rubisco by transforming the nucleus with antisense genes directed at the small subunit. More recently, technology for transforming the small genome of the plastid of tobacco has enabled much more precise manipulation and replacement of the plastome-encoded large subunit. Engineered changes in Rubisco's properties in vivo are reflected as profound changes in the photosynthetic gas-exchange properties of the leaves and the growth requirements of the plants. Unpredictable expression of plastid transgenes and assembly requirements of some foreign Rubiscos that are not satisfied in higher-plant plastids provide challenges for future research.     

Interactions of hydrogen peroxide with ribulose bisphosphate carboxylase oxygenase

Hydrogen peroxide inhibited both carboxylase and oxygenase activities of purified, and fully activated, spinach ribulose-1,5-bisphosphate (RuP2) carboxylase-oxygenase. Inhibition of the carboxylase reaction was mixed competitive with respect to CO2 (Ki = 1.2 mM) and uncompetitive with respect to RuP2. For the oxygenase reaction, H2O2 was a competitive inhibitor with respect to O2 (Ki = 2.1 mM) and an uncompetitive inhibitor with respect to RuP2. H2O2 did not alter the stoichiometry between CO2 and RuP2 in the carboxylase reaction, indicating that H2O2 was not itself a substrate for the enzyme. RuP2 decreased the rate of deactivation of the enzyme which occurred at limiting CO2 concentrations. H2O2 greatly enhanced this stabilizing effect of RuP2 but had no effect on the rate of deactivation in the absence of RuP2. The inhibitory and stabilizing effects of H2O2 varied similarly with H2O2 concentration. These instantaneous, reversible effects of H2O2 were readily distinguishable from an irreversible inhibitory effect which occurred quite slowly, and in the absence of RuP2. These observations are discussed in relation to the enzyme's catalytic mechanism and its activation-deactivation transformations.     

Covalent modification of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodomicrobium vannielii

The most abundant phosphorus-containing polypeptide in the purple non-sulphur bacterium Rhodomic-robium vannielii has been identified by a combination of immunoprecipitation and sucrose density gradient centrifugation as the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase. The covalent modification of the large subunit involves the phosphorylation of one or more tyrosine residues and appears to occur prior to assembly of the large subunit into the mature enzyme. In addition, the phosphorylated form of the large subunit was found to exist in at least two distinct protein complexes of Mr 410,000 and 440,000.     

Characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis Z

An improved method was devised to purify ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) with high specific activity (2.1 mumol of CO2 fixed/mg protein/min) from Euglena gracilis Z. The purified enzyme stored at -80 degrees C required treatment with dithiothreitol for full activity. The dithiothreitol-treated RuBisCO was activated by 12 mM NaHCO3 and 20 mM MgCl2, and the activated state was stable at least for 60 min in the presence of 4 mM ethylenediaminetetraacetate. The form of inorganic carbon fixed by the Euglena enzyme was CO2, as for the plant enzymes. The carboxylase reaction proceeded linearly with time for at least 8 min. The optimum pH for this reaction was 7.8 to 8.0. The carboxylase activity increased with increasing temperature up to 50 degrees C. The activation energy for the carboxylation reaction was 10.0 kcal/mol. The Michaelis constants of Euglena RuBisCO were 30.9 microM for CO2, 560 microM for O2, and 10.5 microM for ribulose 1,5-bisphosphate. Mathematical comparison between the photosynthesis rate predicted from these enzymatic properties and the observed rate suggested that there is no CO2-concentrating mechanism in E. gracilis.     

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.     

The CO2/O2 specificity of ribulose 1,5-bisphosphate carboxylase/oxygenase

The substrate specificity factor, V _cK_o/V_oK_c, of spinach (Spinacia oleracea L.) ribulose 1,5-bisphosphate carboxylase/oxygenase was determined at ribulosebisphosphate concentrations between 0.63 and 200 M, at pH values between 7.4 and 8.9, and at temperatures in the range of 5° C to 40° C. The CO₂/O₂ specificity was the same at all ribulosebisphosphate concentrations and largely independent of pH. With increasing temperature, the specificity decreased from values of about 160 at 5° C to about 50 at 40° C. The primary effects of temperature were on K _c [Km(CO₂)] and V _c [Vmax (CO₂)], which increased by factors of about 10 and 20, respectively, over the temperature range examined. In contrast, K _o [Ki (O₂)] was unchanged and V _o [Vmax (O₂)] increased by a factor of 5 over these temperatures. The CO₂ compensation concentrations () were calculated from specificity values obtained at temperatures between 5° C and 40° C, and were compared with literature values of . Quantitative agreement was found for the calculated and measured values. The observations reported here indicate that the temperature response of ribulose 1,5-bisphosphate carboxylase/oxygenase kinetic parameters accounts for two-thirds of the temperature dependence of the photorespiration/photosynthesis ratio in C₃ plants, with the remaining one-third the consequence of differential temperature effects on the solubilities of CO₂ and O₂.Abbreviations RuBPC/O(ase) ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - CO₂ compensation concentration     

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     

Stoichiometry in the assay of ribulose bisphosphate oxygenase and carboxylase

Complete stoichiometry of the reaction catalyzed by ribulose 1,5-bisphosphate (RuBP) oxygenase from spinach and Rhodospirillum rubrum has been determined. Before initiation and after termination, RuBP has been measured either by release of equimolar orthophosphate at 25°C in the presence of 1 n NaOH or by complete carboxylation using ¹⁴CO₂ and RuBP carboxylase. The RuBP-dependent oxygen consumption has been measured continuously with an oxygen electrode. After termination of catalysis, 3-phosphoglycerate production has been determined spectrophotometrically using phosphoglycerokinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, α-glycerophosphate dehydrogenase, ATP, and NADH. To measure phosphoglycolate, this product was first hydrolyzed with alkaline phosphatase and the resultant glycolate oxidized by glycolate oxidase. Attendant H₂O₂ formation catalyzed by peroxidase has then been measured colorimetrically. Interference by ribulose in the measurement of glycolate can be easily corrected. Procedures are rapid and do not require separation of reactants and products. Results are in excellent accord with the expected stoichiometry for catalysis by RuBP oxygenase and also enable an estimate of competing catalysis by RuBP carboxylase.     

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.