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.     

Isolation of the catalytically competent small subunit of ribulose bisphosphate carboxylase/oxygenase from spinach under an extremely alkaline condition

A method for isolating the small subunit (B) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach leaf using an alkaline buffer (pH 11.2) in combination with sucrose gradient centrifugation is described. Although the yield of isolated subunit B (ca. 20%) was comparable to that previously described (ca. 25%) using the acid precipitation method [Andrews, T.J. and Lorimer, G.H. (1985) J. Biol. Chem. 260: 4632-4636], the isolated subunit B in this report suffered less denaturation (ca. 30%) as estimated from kinetic analysis of its reassembly with large subunit (A) derived from Aphanothece halophytica. Studies on the kinetic properties of the reassembled enzyme molecules suggested that spinach subunit B does not influence the affinity of the enzyme for substrate CO2. The catalytic core (A8) of spinach RuBisCO could not be isolated in the native form.     

Expression of genes for subunits of plant-type RuBisCO from Chromatium and production of the enzymically active molecule in Escherichia coli

A DNA fragment containing genes for both large (A) and small (B) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from a photosynthetic bacterium Chromatium vinosum was ligated with vectors for expressing unfused proteins and introduced into cells of Escherichia coli. The expressers of RuBisCO were screened on agar plates using the specific antibody raised against the native enzyme from Chromatium. The production of both subunits A and B in the expressers was demonstrated by an immunoblotting experiment. The amount of RuBisCO produced in the E. coli cells was as high as 15% of the total soluble protein after induction with isopropyl-beta-D-thiogalactoside. The specific activity of enzyme molecules produced in E. coli was nearly the same as that of the original Chromatium enzyme. On gel filtration high-performance liquid chromatography the two enzymes showed identical elution behavior, strongly indicating their similar quaternary structures.     

Role of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in the activation process

The large (A) and small (B) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from the cyanobacterium Aphanothece halophytica and from the purple sulfur photosynthetic bacterium Chromatium vinosum (strain D) were separated by sucrose density gradient centrifugation at low ionic strength and alkaline pH (9.3), respectively. It was found that subunit B enhances the extent of activation by CO2 and Mg2+ at equilibrium of the two homologous enzymes consisting of Aphanothece large subunit and its own small subunit (AaBa) and the Chromatium large subunit and its own small subunit (AcBc). The extent of activation induced by saturating amounts of subunit B was larger with AcBc than AaBa, amounting to 3.7- and 1.8-fold of that by each catalytic core alone, respectively. Subunit B stimulated both the extent of activation at equilibrium and catalysis in a parallel and simultaneous manner with respect to the concentration of B in both homologous enzymes. These results suggest that subunit B interacts with both activation and catalytic sites simultaneously. On the other hand, Chromatium subunit B only slightly stimulated the extent of activation in the hybrid enzyme AaBc. The role of subunit B in enhancing the extent of activation at equilibrium can be substituted by the effect exerted by 6-phosphogluconate. Both homologous enzymes AaBa and AcBc showed a faster deactivation rate when the enzyme was activated in the absence of subunit B. The mechanism by which subunit B promotes activation seems to involve its effect on stabilizing the activated enzyme molecule. From studies on the Km for substrate CO2 in the hybrid enzyme AaBc a major involvement of subunit B in influencing Km (CO2) seems unlikely.     

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.     

Ribulose 1,5-bisphosphate carboxylase-oxygenase. I. Structural, immunochemical and catalytic properties

Some structural, immunochemical and catalytic properties are examined for ribulose 1,5-bisphosphate carboxylase-oxygenase from various cellular organisms including bacteria, cyanobacteria, algae and higher plants. The native enzyme molecular masses and the subunit polypeptide compositions vary according to enzyme sources. The molecular masses of the large and small subunits from different cellular organisms, on the other hand, show a relatively high homology due to their well-conserved primary amino acid sequence, especially that of the large subunit. In higher plants, the native enzyme and the large subunit are recognized by the antibodies raised against either the native or large subunit, whereas the small subunit apparently cross-reacts only with the antibodies directed against itself. A wide diversity exists, however, in the serological response of the native enzyme and its subunits with antibodies directed against the native enzyme or its subunits from different cellular organisms. According to numerous kinetic studies, the carboxylase and oxygenase reactions of the enzyme with ribulose 1,5-bisphosphate and carbon dioxide or oxygen require activation by carbon dioxide and magnesium prior to catalysis with ribulose 1,5-bisphosphate and carbon dioxide or oxygen. The activation and catalysis are also under the regulation of other metal ions and a number of chloroplastic metabolites. Recent double-labeling experiments using radioactive ribulose 1,5-bisphosphate and 14CO2 have elucidated the carboxylase/oxygenase ratios of the enzymes from different organisms. Another approach, i.e., genetic experiments, has also been used to examine the modification of the carboxylase/oxygenase ratio.     

Heterologous hybridization of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) restores the enzyme activities

The catalytic core (A8) and small subunit (B) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were isolated from two species of cyanobacteria (Aphanothece halophytica and Synechococcus ACMM 323) as well as from the photosynthetic purple sulfur bacterium, Chromatium vinosum. The subunit B is essential for the activity of all three enzymes. The heterologous hybridization of RuBisCO molecules from the three organisms was attempted and the reconstitution of the catalytically active hybrid was achieved between A8 derived from either Aphanothece or Synechococcus and subunit B from Aphanothece, Synechococcus or Chromatium. However, reconstitution of the enzymically active hybrid between A8 from Chromatium and B subunits from the cyanobacteria could not be achieved. Experiments by using high performance liquid column chromatography also showed the formation of a heterologous hybrid possessing RuBP carboxylase activity.     

Phosphorylation in vitro of the large subunit of the ribulose-1,5-bisphosphate carboxylase and of the glyceraldehyde-3-phosphate dehydrogenase

A protein kinase activity responsible for the in vitro phosphorylation of at least six endogenous polypeptides including the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is present in the stroma (3000 X g supernatant, S30) of spinach chloroplasts. The phosphorylation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit is strongly enhanced when sodium fluorure is used as a protein phosphatase inhibitor. Phosphorylation occurs on threonine and serine residues. The protein kinase involved is not Ca2+-dependent. There is also evidence for a protein phosphatase activity which suggests a coupled regulation by a phosphorylation-dephosphorylation process. The phosphorylating activity is drastically reduced when S30 is prepared from leaves harvested after a dark period. Phosphorylation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit is not related to its own synthesis. The in vitro phosphorylation of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) is also demonstrated.     

D-Ribulose-1,5-bisphosphate carboxylase/oxygenase from chemolithotrophically grown Rhizobium japonicum

Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified from chemolithotrophically grown Rhizobium japonicum SR and ribulose-5-phosphate kinase activity has also been detected in extracts of such cells. Electrophoretically homogeneous ribulosebisphosphate carboxylase/oxygenase purified in the presence of PMSF showed two types of large subunits of 55 000 and 53 000 daltons and small subunits of 14 200 daltons. The heterogeneity of large subunits was not observed when the enzyme was prepared in the presence of PMSF and DIFP. Ribulose-1,5-bisphosphate carboxylase from R. japonicum was inhibited by antibodies to this enzyme and a single precipitin band from the antibody-enzyme interaction was observed on double diffusion plates. Antibodies to R. japonicum enzyme did not cross-react on immunodiffusion plates with the ribulosebisphosphate carboxylase/oxygenases from wheat, spinach, soybean and tobacco.     

Further proof for the catalytic role of the larger subunit in the spinach leaf ribulose-1,5-diphosphate carboxylase

The catalytically active oligomeric form of the larger subunit, A_m, obtained from spinach leaf ribulose-1,5-diphosphate carboxylase by pretreatment with p-mercuribenzoate at pH 7.5 followed by incubation at pH 9.0, was free of the smaller subunit based on C-terminal amino acid analyses. Valine was the predominant C-terminus of the A_m preparations, the release of tyrosine being negligibly small [cf. Sugiyama and Akazawa, $\text{Biochemistry}$ 9 (1970) 4499]. The pH optimum of the ribulose-1,5-diphosphate carboxylase reaction by A_m was about 8.5, in comparison to the native enzyme which showed an alkaline pH optimum only in the absence of Mg²⁺. The substrate saturation curve of the catalytic subunit with respect to bicarbonate followed the Michaelis-Menten equation, as contrasted to the anomalous reaction kinetics of the native ribulose-1,5-diphosphate carboxylase molecule reported previously. These overall results indicate that the allosteric properties of spinach ribulose-1,5-diphosphate carboxylase are possibly conveyed by a unique structural conformation that requires the presence of the smaller subunit in association with the larger catalytic subunit component of the enzyme molecule.     

Identification of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase as a Substrate for Transglutaminase in Medicago sativa L. (Alfalfa)

Extracts prepared from floral meristematic tissue of alfalfa (Medicago sativa L.) were investigated for expression of the enzyme transglutaminase in order to identify the major protein substrate for transglutaminase-directed modifications among plant proteins. The large polymorphic subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase in alfalfa, with molecular weights of 52,700 and 57,600, are major substrates for transglutaminase in these extracts. This was established by: (a) covalent conjugation of monodansylcadaverine to the large subunit followed by fluorescent detection in SDS-polyacrylamide gels; (b) covalent conjugation of [¹⁴C]putrescine to the large subunit with detection by autoradiography; (c) covalent conjugation of monodansylcadaverine to the large subunit and demonstration of immunocross-reactivity on nitrocellulose transblot of the modified large subunit with antibody prepared in rabbits against dansylated-ovalbumin; (d) demonstration of a direct dependence of the rate of transglutaminase-mediated, [¹⁴C]putrescine incorporation upon the concentration of ribulose, 1,5-bisphosphate carboxylase/oxygenase from alfalfa or spinach; and (e) presumptive evidence from size exclusion chromatography that transglutaminase may cofractionate with native molecules of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts. Analysis of the primary structure of plant large subunit has revealed numerous potential glutaminyl and lysyl sites for transglutaminase-directed modifications of ribulose 1,5-bisphosphate carboxylase/oxygenase.     

香蕉rbcS基因启动子的克隆及序列分析

以巴西香蕉为材料,根据已经获得的香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的全长cDNA序列设计1对专一引物,通过PCR扩增得到了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基的基因组全长,序列长811 bp,含有2个内含子。根据其基因组序列设计引物,采用SEFA-PCR方法,以总DNA为模板克隆了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的启动子序列,长1 681 bp。用PLACE软件分析发现该序列具有启动子的基本元件TATA-box、CAAT-box,包含多个胁迫诱导元件,如光诱导元件、赤霉素、低温诱导元件、昼夜节律调控元件等。该序列的克隆与分析为进一步研究香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的表达调控奠定了基础。     

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.     

Increased susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase to proteolytic degradation caused by oxidative treatments

The susceptibility of the chloroplastic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase to proteolysis by trypsin, chymotrypsin, proteinase K, and papain is enhanced by oxidative treatments including spontaneous oxidation of cysteines. Proteinases exhibit a high specificity for the oxidized inactive form of the carboxylase, cleaving its large subunit. Treatment of the inactive enzyme with dithiothreitol results in partial recovery of both carboxylase activity and resistance to proteolysis. This behavior may explain the specific degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase that occurs in vivo during leaf senescence.     

Cotranscription, deduced primary structure, and expression of the chloroplast-encoded rbcL and rbcS genes of the marine diatom Cylindrotheca sp. strain N1.

The primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined. Unlike higher plants and green algae, the genes encoding the large and the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase are chloroplast-encoded and closely associated (Hwang and Tabita, 1989). The rbcL and rbcS genes in strain N1 are cotranscribed and are separated by an intergenic region of 46 nucleotide base pairs. Ribosome binding sites and a potential promoter sequence were highly homologous to previously determined chloroplast sequences. Comparison of the deduced primary structure of the diatom large and small subunits indicated significant homology to previously determined sequences from bacteria; there was much less homology to large and small subunits from cyanobacteria, green algae, and higher plants. Although high levels of recombinant diatom large subunits could be expressed in Escherichia coli, the protein synthesized was primarily insoluble and incapable of forming an active hexadecameric enzyme. Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphate carboxylase/oxygenase proteins, despite the distinctive procaryotic gene arrangement and sequence homology.     

Rapid kinetics of an N-terminal mutant of cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase

The transient changes in absorption of visible light upon addition of ribulose 1,5-bisphosphate to Co2(+)-activated ribulose-1,5-bisphosphate carboxylase/oxygenase were used to show altered catalytic properties of a mutant form of the enzyme from Anacystis nidulans. The mutant form of the enzyme had a modified N-terminus and a 10-fold greater Km for ribulose 1,5-bisphosphate than the natural cyanobacterial enzyme.     

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.     

Amino-acid-transport mutant of Nicotiana tabacum L.

The structure of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) has been investigated by tilted-view electron microscopy of negatively stained monolayer crystals and image processing. The structure determined consists of a cylinder of octagonal cross-section with a large central hole. Based on this and other available evidence a model for the arrangement of the large and small subunits is suggested with the eight small subunits arranged equatorially around the core of eight large subunits.Abbreviations LS large subunit - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SS small subunit     

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its precursor expressed in Escherichia coli are associated with groEL protein

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is synthesized in the cytoplasm as a precursor which is transported into the chloroplast. During or after transport the precursor is processed to its mature size by removal of an amino-terminal transit peptide. Eight small subunits and eight large subunits (synthesized in the chloroplast) assemble to form the holoenzyme. We have expressed the precursor of the small subunit in Escherichia coli as a fusion to the carboxyl terminus of staphylococcal protein A'. The fusion protein was recovered from the bacterial lysate by chromatography on IgG-agarose. A 58-kDa protein copurified with the fusion protein in approximately equal amounts. Much less of the 58-kDa protein copurified with a fusion in which the transit peptide was deleted, and it did not copurify with protein A'. The 58-kDa protein was identified as the E. coli groEL gene product with antibodies directed against a homologous mitochondrial heat shock protein. This finding is particularly interesting because a chloroplast protein involved in the assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase also is homologous to the groEL protein. These homologs could modulate protein-protein interactions during folding and assembly of subunits into native complexes.     

Exchange Properties of the Activator CO(2) of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

The exchange properties of the activator CO₂ of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO₂ on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO₂. The exchange kinetics of the activator CO₂ were monitored by activation of the enzyme to steady state in the presence of ¹²CO₂, followed by addition of ¹⁴CO₂ and determination of the amount of labeled CO₂ trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K_obs) for exchange with both the purified enzyme (0.45 min⁻¹) and in illuminated chloroplasts (0.18 min⁻¹) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO₂ was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.