Ribulose diphosphate carboxylase/oxygenase. III. Isolation and properties.

Similarities in properties of ribulose diphosphate carboxylase and oxygenase activities further substantiate the hypothesis that the same protein catalyzes both reactions. The Km (ribulose diphosphate) is 0.33 mM for the ribulose diphosphate oxygenase, when assayed in air with an oxygen electrode. Maximum activity is obtained with 10 to 35 mM MgCl2. Higher MgCl2 concentrations are inhibitory, but they shift the pH optimum from 9.3 or 9.4 to 8.7 or 9.0. MnCl2 is an effective cofactor of the oxygenase and some activity is obtained with CoCl2. Both the ribulose diphosphate carboxylase and oxygenase activity of the purified protein from spinach leaves are slowly inactivated by storage at 0 degrees and reactivated in 10 min at 50 degrees, provided both 25 mM MgCl2 and 1 mM dithiothreitol are present. The sulfhydryl groups of the enzyme which react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) are approximately 4 at pH 7.8 and 11 at pH 9.4. At both pH values ribulose diphosphate prevents two of these sulfhydryl groups from reacting with this reagent. About 50% inhibition of the oxygenase activity at pH 9.0 occurs with 50 mM bicarbonate in the presence of 3 mM ribulose diphosphate, and from variations in these parameters the inhibition is attributed to the CO2 species. The purified enzyme of acrylamide gels prevented the reduction of nitroblue tetrazolium in the presence of the superoxide radical, but the enzyme in solution did not react as a superoxide dismutase.     

Ribulose diphosphate oxygenase. V. Presence in ribulose diphosphate carboxylase from Rhodospirillum rubrum

Ribulose-1,5-diphosphate carboxylase was purified fifteenfold from Rhodospirillum rubrum grown autotrophically under H₂ and CO₂. There was RuDP oxygenase activity associated with the carboxylase. The oxygenase had maximal activity at pH 9.4. Although these bacterial RuDP oxygenase and carboxylase activities were cold labile, activity could not be restored by treatment at 50° in the presence of Mg⁺⁺ and a sulfhydryl reagent, in contrast to results with the enzyme from eukaryotes.     

Ribulose bisphosphate carboxylase/oxygenase from Thiocapsa roseopersicina

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified 80-fold from malate-grown Thiocapsa roseopersicina by salting out the enzyme from the high-speed supernatant between 68–95% saturation with respect to (NH₄)₂SO₄, gelfiltration through Sephadex G-100, and DEAE-cellulose chromatography followed by sedimentation into a 14–34% glycerol gradient. The specific activity of enzyme for the carboxylase reaction was 2.45 mol RuBP-dependent CO₂ fixed/min · mg protein (at pH 8.0 and 30° C) and for the oxygenase reaction was 0.23 mol RuBP-dependent O₂ consumed/min · mg protein (at pH 8.6, and 25° C). The enzyme, which was ultracentrifugally homogeneous in the presence of 4 and 10% v/v glycerol, was stable for at least one year at-80° C in the presence of 10% glycerol. S_{20, w} values obtained in the presence of 4 and 10% glycerol were 19.3 and 16.2, respectively. The enzyme contained both large (53,000-daltons) and mixed small subunits (15,000- and 13,500-daltons).Borate-dependent inactivation of the enzyme by 2,3-butadione, which was greatly reduced in the presence of the product 3-phosphoglycerate, suggested that one or more arginines are at the active site.Abbreviations DTT dithiotreitol - RuBP d-ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate - TCA trichloroacetic acid - TEMBDG buffer (pH 8.0 at 25°C) containing 20 mM Tris, 1 mM disodium EDTA · 2 H₂O, 10 mM MgCl₂·6 H₂O, 50 mM NaHCO₃, 0.1 mM DTT and 10% glycerol (v/v)     

Ribulose diphosphate carboxylase from autotrophic microorganisms

Thiobacillus denitrificans was grown anaerobically with nitrate as an acceptor in both sterile and nonsterile media. Ribulose diphosphate carboxylase was stable throughout the exponential growth phase and declined slowly only after cells reached the stationary phase. Reversible inactivation of the carboxylase occurred in extracts as a result of bicarbonate omission. The enzyme was purified 32-fold with excellent recovery of a preparation which was 50 to 60% pure by the criterion of polyacrylamide gel electrophoresis. This purified preparation catalyzed the fixation of 1.25 mumoles of CO(2) per min per mg of protein at pH 8.1 and 30 C, and the molecular weight of ribulose diphosphate carboxylase was approximately 350,000 daltons. A striking biphasic time course of CO(2) fixation that was independent of protein and ribulose diphosphate concentration was observed. The optimal pH of the enzyme assay was fairly broad, ranging from 7 to 8.2. Kinetic dependence upon bicarbonate, ribulose diphosphate, and Mg(2+) was characterized and indicated that bicarbonate and Mg(2+) must combine with enzyme prior to addition of ribulose diphosphate. Antiserum to ribulose diphosphate carboxylase from Hydrogenomonas eutropha was only slightly inhibitory when added to the enzyme from T. denitrificans, and the mixture did not precipitate. Cyanide (4 x 10(-5)m) gave 61% inhibition of the enzyme from T. denitrificans. Ribulose diphosphate carboxylase in extracts of H. eutropha, H. facilis, Chromatium D, Rhodospirillum rubrum, and Chlorella pyrenoidosa were also inhibited to varying extents by cyanide and antiserum to the H. eutropha enzyme.     

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.     

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%.     

Ribulose bisphosphate carboxylase/oxygenase content determined with [C]carboxypentitol bisphosphate in plants and algae

As is the case with spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco), [¹⁴C]carboxyarabinitol bisphosphate (CABP) bound to purified Chlorella Rubisco with a molar ratio of unity to large subunit of the enzyme. The concentration of binding sites in extracts of photosynthetic organisms was determined by reacting the extracts with [¹⁴C]-carboxypentitol bisphosphate (CPBP) and precipitating the resultant Rubisco-[¹⁴C]CABP complex with a combination of polyethylene glycol-4000 and MgCl₂. Plots of the relationship between concentrations of [¹⁴C] CPBP in the reaction mixture and the precipitated [¹⁴C]CPBP gave a straight line and the concentration of binding sites were estimated by extrapolation to zero [¹⁴C]CPBP since the dissociation constant of CABP with Rubisco is 10⁻¹¹ molar. Spinach, pea, and soybean leaves contained 6.4 to 6.8 milligrams Rubisco per milligram chlorophyll, corresponding to 92 to 97 ribulose bisphosphate-binding sites per milligram chlorophyll. The Rubisco content of sunflower and wheat leaves was 5.3 to 5.5 milligrams per milligram chlorophyll. The concentrations in C₄ plants were not uniform and corn and Panicum miliaceum leaves contained 3 and 7 milligrams Rubisco per milligram chlorophyll. The Rubisco content of green algae was one-fifth to one-sixth that of C₃ plant leaves and was affected by the CO₂ concentration during growth. The content of Euglena and blue-green algae is also reported.     

Regeneration of Ribulose 1,5-bisphosphate and Ribulose 1,5-bisphosphate carboxylase/oxygenase Activity Associated with Lack of Oxygen Inhibition of Photosynthesis at Low Temperature

The nature of the lack of oxygen inhibition of C₃-photosynthesisat low temperature was investigated in white clover (Trifoliumrepens L.). Detached leaves were brought to steady-state photosynthesisin air (34 Pa p(CO²), 21 kPa p(O₂), balance N₂) at temperaturesof 20°C and 8°C, respectively. Net photosynthesis, ribulose1,5-bisphosphate (RuBP) and ATP contents, and ribulose 1,5-bisphosphatecarboxylase/oxygenase (RuBPCO) activities were followed beforeand after changing to 2·0 kPa p(O₂). At 20°C, lowering p(O₂) increased net photosynthesis by37%. This increase corresponded closely with the increase expectedfrom the effect on the kinetic properties of RuBPCO. Conversely,at 8°C net photosynthesis rapidly decreased following adecrease in p(O₂) and then increased again reaching a steady-statelevel which was only 7% higher than at 21 kPa p(O₂). The steady-staterates of RuBP and associated ATP consumption were both estimatedto have decreased. ATP and RuBP contents decreased by 18% and33% respectively, immediately after the change in p(O₂) suggestingthat RuBP regeneration was reduced at low p(O₂) due to reducedphotophosphorylation. Subsequently, RuBP content increased again.Steady-state RuBP content at 2·0 kPa p(O₂) was 24% higherthan at 21 kPa p(O₂). RuBPCO activity decreased by 22%, indicatingcontrol of steady-state RuBP consumption by RuBPCO activity. It is suggested that lack of oxygen inhibition of photosynthesisat low temperature is due to decreased photophosphorylationat low temperature and low p(O₂). This may be due to assimilateaccumulation within the chloroplasts. Decreased photophosphorylationseems to decrease RuBP synthesis and RuBPCO activity, possiblydue to an acidification of the chloroplast stroma. Key words: Oxygen inhibition, photosynthesis, ribulose bisphosphate carboxylase/oxygenase     

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.     

Pyruvate is a by-product of catalysis by ribulosebisphosphate carboxylase/oxygenase

Pyruvate is a minor product of the reaction catalyzed by ribulosebisphosphate carboxylase/oxygenase from spinach leaves. Labeled pyruvate was detected, in addition to the major labeled product, 3-phosphoglycerate, when 14CO2 was the substrate. Pyruvate production was also measured spectrophotometrically in the presence of lactate dehydrogenase and NADH. The Km for CO2 of the pyruvate-producing activity was 12.5 microM, similar to the CO2 affinity of the 3-phosphoglycerate-producing activity. No pyruvate was detected by the coupled assay when ribulose 1,5-bisphosphate was replaced by 3-phosphoglycerate or when the carboxylase was inhibited by the reaction-intermediate analog, 2'-carboxyarabinitol 1,5-bisphosphate. Therefore, pyruvate was not being produced from 3-phosphoglycerate by contaminant enzymes. The ratio of pyruvate produced to ribulose bisphosphate consumed at 25 degrees C was 0.7%, and this ratio was not altered by varying pH or CO2 concentration or by substituting Mn2+ for Mg2+ as the catalytically essential metal. The ratio increased with increasing temperature. Ribulose-bisphosphate carboxylases from the cyanobacterium Synechococcus PCC 6301 and the bacterium Rhodospirillum rubrum also catalyzed pyruvate formation and to the same extent as the spinach enzyme. When the reaction was carried out in 2H2O, the spinach carboxylase increased the proportion of its product partitioned to pyruvate to 2.2%. These observations provide evidence that the C-2 carbanion form of 3-phosphoglycerate is an intermediate in the catalytic sequence of ribulose-bisphosphate carboxylase. Pyruvate is formed by beta elimination of a phosphate ion from a small portion of this intermediate.     

Vitamin K-dependent oxygenase/carboxylase; differential inactivation by sulfhydryl reagents

Inhibition of vitamin K-dependent carboxylase and oxygenase by sulfhydryl reagents was compared. Formation of vitamin K epoxide and vitamin K-dependent carboxylation are both strongly (greater than 90%) inhibited by l mM p-hydroxy-mercuribenzoate, and this inhibition is reversed by dithiothreitol. Both activities are also effectively inhibited by N-ethylmaleimide (NEM). Preincubation with vitamin K hydroquinone prevents NEM inhibition of epoxide formation but not of carboxylation. These data argue that separate active sites are required to support vitamin K-dependent epoxide formation and carboxylation and that the binding site vitamin K oxygenase contains an active thiol group.     

Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit translation is regulated in a small subunit-independent manner in the expanded leaves of tobacco

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is composed of small subunits (SSs) encoded by rbcS on the nuclear genome and large subunits (LSs) encoded by rbcL on the chloroplast genome, and it is localized in the chloroplast stroma. Constitutive knockdown of the rbcS gene reportedly causes a reduction in LS quantity and the level of translation in tobacco and the unicellular green alga Chlamydomonas. Constitutively knockdown of the rbcS gene also causes a reduction in photosynthesis, which influences the expression of photosynthetic genes, including the rbcL gene. Here, to investigate the influence of the knockdown of the rbcS gene on the expression of the rbcL gene under normal photosynthetic conditions, we generated transgenic tobacco plants in which the amount of endogenous rbcS mRNA can be reduced by inducible expression of antisense rbcS mRNA with dexamethasone (DEX) treatment at later stages of growth. In already expanded leaves, after DEX treatment, the level of photosynthesis, RuBisCO quantity and the chloroplast ultrastructure were normal, but the amount of rbcS mRNA was reduced. An in vivo pulse labeling experiment and polysome analysis showed that LSs were translated at the same rate as in wild-type leaves. On the other hand, in newly emerging leaves, the rbcS mRNA quantity, the level of photosynthesis and the quantity of RuBisCO were reduced, and chloroplasts failed to develop. In these leaves, the level of LS translation was inhibited, as previously described. These results suggest that LS translation is regulated in an SS-independent manner in expanded leaves under normal photosynthetic conditions.