Catalytic role of subunit A in ribulose-1,5-diphosphate carboxylase from Chromatium strain D

The oligomeric form of the larger subunit designated as A_m produced by alkali treatment of ribulose-1,5-diphosphate carboxylase from the purple sulfur bacterium, Chromatium strain D, retained partial enzymic activity in the absence of the small subunit (B). Supporting evidence was obtained by polyacrylamide gel electrophoresis at pH 8.9 and Sephadex G-200 gel filtration equilibrated with alkaline buffer at pH 9.2. The specific enzyme activity of A_m (45 nmoles CO₂ fixed/mg protein/min) was approximately 15% of the native intact enzyme molecule. By sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the A_m preparation was proved to be free from contamination of subunit B. With reservation of the sensitivity limit of this particular technique we concur that the larger subunit is the catalytic entity of the carboxylase reaction. The optimum pH of the ribulose-1,5-diphosphate carboxylase reaction catalyzed by isolated A_m lies on the alkaline side at about pH 8.3 with or without Mg²⁺. The undissociated native enzyme possesses an optimum pH on the alkaline side in the absence of Mg²⁺, which shifts to the acidic side in the presence of Mg²⁺. From this behavior it is inferred that the association of the smaller subunit with the larger subunit causes conformational stabilization of the enzyme molecule with an accompanying change in the pH optimum due to Mg²⁺.     

Some properties of coupled hepatoma mitochondria exhibiting uncoupler-insensitive ATPase activity

Ribulose-1,5-diphosphate car?ylase from the photosynthetic bacterium Chromatium catalyses the oxidative formation of phosphoglycolate and 3-phosphoglycerate from ribulose-1,5-diphosphate at an alkaline pH (9.3) in an atmosphere of oxygen. The catalytically active oligomeric form of the large subunit of the car?ylase molecule, A_m, was proved to be functionally active in the ribulose-1,5-diphosphate oxygenase reaction without the presence of the smaller subunit.     

Localization and properties of ribulose diphosphate carboxylase from castor bean endosperm

A substantial portion of the ribulose 1,5-diphosphate carboxylase activity in the endosperm of germinating castor beans (Ricinus communis var. Hale) is recovered in the proplastid fraction. The partially purified enzyme shows homology with the enzyme from spinach (Spinacia oleracea) leaves, as evidenced by its reaction against antibodies to the native spinach enzyme and to its catalytic subunit. The enzyme from the endosperm of castor beans has a molecular weight of about 500,000 and, with the exception of a higher affinity for ribulose 1,5-diphosphate, has similar kinetic properties to the spinach enzyme. The castor bean carboxylase is inhibited by oxygen and also displays ribulose 1,5-diphosphate oxygenase activity with an optimum at pH 7.5.     

Further studies on the subunit structure of Chromatium ribulose-1,5-phosphate carboxylase.

Upon alkali exposure Chromatium ribulose-1,5-bisphosphate carboxylase dissociates into constituent subunits, a catalytic oligomer of the larger subunit, A8, and monomeric form of the small subunit B. By sedimentation equilibrium molecular weights of the native enzyme and the catalytic oligomer produced by an alkali treatment were estimated to be 5.11 x 10 5 and 4.29 x 10 5, respectively. To provide information on reversibility of the dissociation by determining whether the enzymically inactive small subunit B of the whole enzyme molecule did indeed exchange with exogenously added subunit B a radioisotopic method was used. After initial alkaline dialysis at pH 9.2 of a mixture of a nonlabeled native enzyme preparation and 14C-labeled subunit B, and the subsequent dialysis at pH 7.0, incorporation of 14C into the recovered native enzyme was determined. Without the alkaline treatment there was no detectable exchange, while after alkaline dialysis for 5 and 10 hr the subunit B exchange was 89 and 82%, respectively. Rabbit antiserum prepared against the catalytic oligomer of the spinach ribulose-1,5-bisphosphate carboxylase, anti-(A) (spinach), inhibited the Chromatium carboxylase and oxygenase activities. This result together with the identical immunoprecipitation lines on an agar plate formed between the antiserum and the Chromatium carboxylase and between the antiserum and the catalytic subunit of the Chromatium enzyme strongly indicated structural near identity of the catalytic subunits of the spinach and Chromatium carboxylase molecules. Results also show that the catalytic site of the Chromatium ribulose-1,5-bisphosphate carboxylase and oxygenase exists in the large polypeptide chain.     

Ribulose-1, 5-bisphosphate carboxylase from comfrey: Isolation and characterization

Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified to electrophoretic homogeneity from comfrey, Symphytum spp. Sodium dodecyl sulfate polyacrylamide and polyacrylamide gel electrophoresis studies on the purified product showed no extraneous proteins. Comparisons of the electrophoretic mobilities of the subunits to those of standard proteins indicated a large subunit MW of 50 000 and a small subunit of 12 700, which for an octameric structure of each subunit indicates a native MW of 502 000. Specific activities of the comfrey enzyme ranged from 1.2 to nearly 2 μmol ¹⁴CO₂ fixed/min.mg of protein over several preparations and were maintained for months when stored from the sucrose gradient at ? 70°. The specific activities depended critically on the amounts of enzyme used in the assay even under saturating conditions of substrates and cofactors. The effective pH dependence for carboxylase catalysis peaked near 7.4, which apparently is the lowest elective optimum yet reported for this enzyme from any source. However, on a constant carbon dioxide basis the pH dependence profile was reversed with a maximum near pH 8.6 which was 0.4 units higher than the value for the spinach enzyme. The K_ms for carbon dioxide and ribulose-1,5-bisphosphate at pH 7.5 were 130 μM and 30 μM, respectively, which are comparable to the accepted values for the carboxylase from spinach at pH 7.2.     

Enzyme levels in relation to obligate phototrophy in chlamydobotrys

During the transition from photoheterotrophic growth on acetate to phototrophic growth on carbon dioxide, there is a decrease in isocitrate lyase and increase in ribulose-1,5-diphosphate carboxylase activity in Chlamydobotrys stellata cultures. The increase in ribulose-1,5-diphosphate carboxylase activity is the result of protein synthesis, there being a close correlation between increase in enzyme activity and protein precipitated by antibody to ribulose-1,5-diphosphate carboxylase. The purified ribulose-1,5-diphosphate carboxylase was similar to the constitutive enzyme from other green algae having a molecular weight of 530,000 and composed of two types of subunit of molecular weight 53,000 and 14,000.     

Properties of ribulose diphosphate carboxylase immobilized on porous glass

Ribulose-1,5-diphosphate carboxylase from spinach has been bound to arylamine porous glass with a diazo linkage and to alklamine porous glass with glutaraldehyde. Stability at elevated temperatures and responses to changes of pH and ribulose-1,5-diphosphate, Mg²⁺, and dithiothreitol concentrations were not significantly different from the soluble enzyme, though stability at 4°C was somewhat improved.     

Inactivation of ribulosebisphosphate carboxylase/oxygenase from rhodospirillum rubrum and spinach with the new affinity label 2-bromo-1,5-dihydroxy-3-pentanone 1,5-bisphosphate

In an attempt to identify the active-site base believed to initiate catalysis by ribulosebisphosphate carboxylase, we have synthesized 2-bromo-1, 5-dihydroxy-3-pentanone 1,5-bisphosphate, a reactive analogue of a postulated intermediate of carboxylation. Although highly unstable, this compound can be shown to inactivate the carboxylases from both $\text{Rhodospirillum}\text{,}\text{rubrum}$ and spinach rapidly and irreversibly. Inactivation follows pseudo first-order kinetics, shows rate saturation and is greatly reduced by saturating amounts of the competitive inhibitor, 2-carboxyribitol 1,5-bisphosphate. The incorporation of reagent, quantified by reducing the modified carboxylases with [³H]NaBH₄, shows that inactivation results from the modification of approximately one residue per catalytic subunit of the $\text{Rhodospirillum}\text{,}\text{rubrum}$ enzyme and less than one residue per protomeric unit of the spinach enzyme.     

The role of sulfhydryl groups in the ribulose- 1,5-bisphosphate carboxylase and oxygenase reactions.

Ribulose-l,5-bisphosphate carboxylase (E.C. 4.1.1.39) isolated from Chromatium strain D contains 64 free cysteinyl -SH groups per mol (M_r 5.11 × 10⁵) as determined using three different titrants: p-[¹⁴C]chloromercuribenzoate, the Ellman reagent, and [¹⁴C]iodoacetamide.Distribution of -SH groups in the two constituent subunits (A and B) isolated from spinach and Chromatium ribulose-1,5-bisphosphate carboxylases was determined to be for spinach, 9 in A and 3 in B; and for Chromatium, 7 in A and 1 in B.The relationship between the numbers of -SH groups blocked vs residual activities of both the ribulose-1,5-bisphosphate carboxylase and oxygenase reactions was examined by titration with p-chloromercuribenzoate. In both spinach and Chromatium enzymes, antisigmoidal curves were obtained for the degree of the enzyme activity loss in relation to the numbers of -SH groups masked. However, at alkaline pH the Chromatium enzyme shows a sharp decline in both carboxylase and oxygenase activities, apparently due to the alkali dissociation of the enzyme molecule accompanied by its structural deformation. The functional role of -SH groups in the ribulose-1,5-bisphosphate carboxylase molecule is discussed in relation to two constituent enzyme reactions, and it is concluded that in both enzyme sources the active sites are probably the same for the two reactions.     

The structure of form I crystals of D-ribulose-1,5-diphosphate carboxylase.

Single crystals of d-ribulose-1,5-diphosphate carboxylase from tobacco leaves, Nicotiana tabacum (variety Turkish Samsun), have been examined by X-ray diffraction, electron microscopy, and optical diffraction. Twelve molecules are loosely packed into a body-centered cubic unit cell, space group I4¹32 with cell dimension $\text{a = 383}\text{A}\text{̊}$. The asymmetric unit is one quarter of a molecule, and the minimum molecular symmetry is 222. This symmetry when combined with estimates of the two subunit masses and stoichiometry is compatible with a molecular structure of the composition L₈S₈ (L is large subunit, S is small). If all bonds between large and small subunits are equivalent, the true molecular symmetry is 422; this symmetry is consistent with molecular images in micrographs.     

The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl₂ followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose- 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay.     

Protein composition of the carboxysomes of Thiobacillus neapolitanus

For purifying carboxysomes of Thiobacillus neapolitanus an isolation procedure was developed which resulted in carboxysomes free from whole cells, protoplasts and cell fragments. These purified carboxysomes are composed of 8 proteins and at the most of 13 polypeptides. The two most abundant proteins which make up more than 60% of the carboxysomes, are ribulose-1,5-bisphosphate carboxylase and a glycoprotein with a molecular weight of 54,000. The shell of the carboxysomes consists of four glycoproteins, one also with a molecular weight of 54,000. The other proteins are present in minor quantities. Ribulose-1,5-bisphosphate carboxylase is the only enzyme which could be detected in the carboxysomes and 3-phosphoglycerate was the only product formed during incubation with ribulose-1,5-diphosphate and bicarbonate. The supernatant of a broken and centrifuged carboxysome suspension contained the large subunit of ribulose-1,5-bisphosphate carboxylase. The small subunit of ribulose-1,5-bisphosphate carboxylase was found in the pellet together with the shell proteins which indicates that the small subunit of ribulose-1,5-bisphosphate carboxylase is connected to the shell.Abbreviations RuBisCO ribulose-1,5-bisphosphate carboxylase - PMSF phenylmethylsulfonyl fluoride - PAA gelectrophoresis, polyacrylamide gelelectrophoresis - SDS sodium dodecyl sulphate - CIE crossed immunoelectrophoresis - IEF isoelectric focusing     

Partial Purification and Characterization of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit epsilonN-Methyltransferase

The large subunit (LS) of tobacco (Nicotiana rustica) ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P₂ carboxylase) contains a trimethyllysyl residue at position 14, whereas this position is unmodified in spinach ribulose-P₂ carboxylase. A protein fraction was isolated from tobacco chloroplasts by rate-zonal centrifugation and anion-exchange fast protein liquid chromatography that catalyzed transfer of methyl groups from S-adenosyl-[methyl-³H]-l-methionine to spinach ribulose-P₂ carboxylase. ³H-Methyl groups incorporated into spinach ribulose-P₂ carboxylase were alkaline stable but could be removed by limited tryptic proteolysis. Reverse-phase high-performance liquid chromatography of the tryptic peptides released after proteolysis showed that the penultimate N-terminal peptide from the LS of spinach ribulose-P₂ carboxylase contained the site of methylation, which was identified as lysine-14. Thus, the methyltransferase activity can be attributed to S-adenosylmethionine:ribulose-P₂ carboxylase LS (lysine) `N-methyltransferase, a previously undescribed chloroplast enzyme. The partially purified enzyme was specific for ribulose-P₂ carboxylase and exhibited apparent K_m values of 10 micromolar for S-adenosyl-l-methionine and 18 micromolar for ribulose-P₂ carboxylase, a V_max of 700 picomoles CH₃ groups transferred per minute per milligram protein, and a broad pH optimum from 8.5 to 10.0. S-Adenosylmethionine:ribulose-P₂ carboxylase LS (lysine)^εN-methyltransferase was capable of incorporating 24 ³H-methyl groups per spinach ribulose-P₂ carboxylase holoenzyme, forming 1 mole of trimethyllysine per mole of ribulose-P₂ carboxylase LS, but was inactive on ribulose-P₂ carboxylases that contain a trimethyllysyl residue at position 14 in the LS. The enzyme did not distinguish between activated (Mg²⁺ and CO₂) and unactivated forms of ribulose-P₂ carboxylase as substrates. However, complexes of activated ribulose-P₂ carboxylase with the reaction-intermediate analogue 2′-carboxy-d-arabinitol-1,5-bisphosphate, or unactivated spinach ribulose-P₂ carboxylase with ribulose-1,5-bisphosphate, were poor substrates for tobacco LS ^εN-methyltransferase.     

Small-angle X-ray scattering of D-Ribulose-1,5-diphosphate carboxylase from Dasycladus clavaeformis roth (Ag.) in solution

D-Ribulose-1,5-diphosphate carboxylase from $\text{Dasycladus}$ was purified, and the gross dimensions were obtained by means of small-angle X-ray scattering measurements in solution. Dissolved single crystals of this enzyme (called “fraction I protein”) gave the same hydrodynamic parameters as the purified form. The molecular weight was found to be 535,000, and a radius of gyration of R_g = 45.5 Å was determined. The experimental scattering curves revealed a geometrical particle of D-Ribulose-1,5-diphosphate carboxylase with gross dimensions of that of a hollow sphere with outer radius of 56 Å and inner radius of 12 Å. Determinations of the diffusion coefficients lead to the conclusion that the enzyme has a spherical shape of almost uniform density.     

Homotropic effect of CO 2 in ribulose-1,5-diphosphate carboxylase reaction

The concentration effect of aqueous CO₂ on the reaction velocity of spinach leaf ribulose-1,5-diphosphate carboxylase has been reevaluated. The homotropic effect of CO₂ in the enzyme reaction supports the previously reported allosteric nature of the enzyme in the CO₂-fixation process in chloroplasts. The concentration of CO₂ giving the half maximal reaction velocity, S_0.5, has been calculated to be 1.47 × 10⁻⁵M.     

Antimycin A Stimulation of Rate-limiting Steps of Photosynthesis in Isolated Spinach Chloroplasts

Changes in levels of metabolites in isolated spinach (Spinacia oleracea) chloroplasts seen upon addition of antimycin A suggest that the activities of enzymes mediating several regulated reactions are affected. Apparently, the presence of added antimycin A does not increase the level of CO₂ in the chloroplasts, nor does it stimulate CO₂ fixation by increasing the level of the carboxylation substrate, ribulose-1,5-diphosphate. Rather, it appears that antimycin A increases CO₂ fixation rate by indirectly stimulating the enzyme, ribulose-1,5-diphosphate carboxylase (E.C. 4.1.1.39), which mediates the carboxylation of ribulose-1,5-diphosphate to give 3-phosphoglycerate. Another rate-limiting enzyme of the reductive pentose phosphate cycle, hexose diphosphatase (E.C. 3.1.3.11), seems also to be stimulated. The synthesis of polysaccharides (mostly starch) seems also to be stimulated. These results are interpreted as indicating that antimycin A addition enhances the general activation of those enzymes which already are activated during photosynthesis but are inactive in the dark. The ratio of adenosine triphosphate-adenosine diphosphate under conditions of photosynthesis was only moderately decreased in the presence of antimycin A, perhaps accounting in part for an observed increase in accumulation of 3-phosphoglycerate as compared with dihydroxyacetone phosphate. No significant effect on movement of metabolites from the chloroplast to the medium was seen.     

The effect of SO 3 -- on the activity of ribulose-1,5-diphosphate carboxylase in isolated spinach chloroplasts

Summary SO ₃ ^-- inhibits the activity of ribulose-1,5-diphosphate carboxylase in isolated spinach chloroplasts. It shows a non-competitive inhibition pattern with respect to ribulose-1,5-diphosphate and Mg⁺⁺ but a competitive one with respect to HCO ₃ ^- . The K _i -values are 14 mM SO ₃ ^-- and 9.5 mM SO ₃ ^- respectively for the non-competitive inhibition but only 3.0 mM SO ₃ ^-- in the case of competitive inhibition with HCO ₃ ^-- as a substrate. Thus it is concluded that the competitive inhibition type will predominate at low SO ₃ ^-- and low internal CO₂ concentrations.The abbreviations used RuDph ribulose-1,5-diphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetate     

The Activity of Ribulose Diphosphate Carboxylase in Extracts of Gonyaulax polyedra in the Day and the Night Phases of the Circadian Rhythm of Photosynthesis

The ribulose 1,5-diphosphate carboxylase from Gonyaulax polyedra Stein. has a half-life of about four hours in buffer, but can be stabilized by the addition of 50% glycerol. The optimum pH is 7.8 to 8.0 and the optimum Mg²⁺ concentration is 3 mm. Heavy metal ions (Cu²⁺, Hg²⁺, Ni²⁺, Zn²⁺), EDTA, pyrophosphate, and adenosine triphosphate were strongly inhibitory. Ribulose 1,5-diphosphate carboxylase from Gonyaulax was not cold-sensitive or activated by light activation factor from tomato or Gonyaulax. No difference in the activity of this enzyme was detected when extracts prepared at the maximum and the minimum of the circadian rhythm of photosynthesis were compared. The Km of HCO₃⁻ was also the same (16 to 19 mm).     

Specific inhibition of oxygenase activity of ribulose-1,5-diphosphate carboxylase by hydroxylamine

Ribulose-1,5-diphosphate oxygenase activity of ribulose-1,5-diphosphate carboxylase was completely inhibited by preincubation of the enzyme with 5mM hydroxylamine in presence of the substrate ribulose-1,5-diphosphate. Inhibition by hydroxylamine was uncompetitive with respect to ribulose-1,5-diphosphate and noncompetitive with respect to magnesium. Carboxylase activity was not affected by hydroxylamine. These results suggest that the two activities of the enzyme can be regulated differentially and that inhibiting the oxygenase activity does not stimulate the carboxylase activity of the enzyme. The data further suggest that the inhibition by hydroxylamine may be through its interaction with carbonyl groups of the enzyme exposed on the binding of ribulose-1,5-diphosphate to the protein.     

Heterogeneity of large subunits of ribulose-1,5-bisphosphate carboxylase from Hydrogenomonas eutropha

Homogeneous ribulose-1,5-bisphosphate carboxylase purified from autotrophically grown $\text{Hydrogenomonas eutropha}$ can be dissociated with sodium dodecylsulfate into small 15,000-dalton subunits and large 56,000- and 52,000-dalton subunits (the latter in a mole ratio of 5:3). The overal mole ratio of small to large subunits is 1.08. Considering the molecular weight of the native enzyme (516,000), the simplest quarternary structure of this enzyme consists of 8 large (mixed) and 8 small subunits. Isolation of the enzyme from cells under conditions that should minimize proteolysis has no effect upon the observed heterogeneity of the large subunits.