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.     

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.     

Essentiality of the small subunit (B) in the catalysis of RuBP carboxylase/oxygenase is not related to substrate-binding in the large subunit (A)

The small subunit (B) of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase from Aphanothece halophytica is absolutely required for the catalysis, but depletion of subunit B does not significantly affect the formation of the quaternary complex-[enzyme.activator CO2.Mg.carboxyarabinitol bisphosphate] in the catalytic core. The inhibition of RuBP carboxylase activity by the reaction of the epsilon-amino group of a lysine in the RuBP-binding site with pyridoxal 5-P is the same whether subunit B is added to the catalytic core before or after the inactivating reaction. The function of subunit B is not related to the substrate binding.     

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.     

Subunit dissociation and reconstitution of ribulose-1,5-bisphosphate carboxylase from Chromatium vinosum

The large and small subunits of ribulose bisphosphate carboxylase from Chromatium vinosum were dissociated and separated at pH 9.6 by sucrose density gradient centrifugation. After further purification by gel filtration, the small subunit fraction contained no carboxylase activity. The large subunit fraction was highly depleted of small subunit based on analysis by denaturing polyacrylamide gel electrophoresis. Carboxylase activity of the large subunit fraction was approximately 1% of the untreated native enzyme. Addition of purified small subunit to the large subunit fraction yielded increases of up to 67-fold in carboxylase activity, further indicating that both subunit types are required for catalysis by this enzyme. The isolated large subunit was fully capable of high-affinity activator 14CO2 binding in the presence of Mg2+ and 2-carboxyarabinitol bisphosphate, indicating that the activator and catalytic sites were not grossly denatured by the depletion of small subunit. Kinetic constants of the native C. vinosum enzyme defined a new class of ribulose bisphosphate carboxylase, which permits the detection of possible kinetic differences if the large and small subunits can be favorably reassembled with those of another kinetic class. From experiments with the enzymes from tobacco and spinach leaves it is concluded that the enzyme from higher plant sources is not suitable for such dissociation/reconstitution-type experiments.     

Molecular and catalytic properties of ribulose 1,5-bisphosphate carboxylase from the photosynthetic extreme halophile Ectothiorhodospira halophila.

D-Ribulose 1,5-bisphosphate (RuBP) carboxylase has been purified from the photosynthetic extreme halophile Ectothiorhodospira halophila. Despite a growth requirement for almost saturating sodium chloride in the medium, both crude and homogeneous preparations of RuBP carboxylase obtained from this organism were inhibited by salts. Sedimentation equilibrium analyses showed the enzyme to be large (molecular weight: 601,000). The protein was composed of two types of polypeptide chains of 56,000 and of 18,000 daltons. The small subunit appeared to be considerably larger than the small subunit obtained from the RuBP carboxylase isolated from Chromatium, an organism related to E. halophila. Amino acid analyses of hydrolysates of both E. halophilia and Chromatium RuBP carboxylases were very similar. Initial velocity experiments showed that the E. halophila RuBP carboxylase had a Km for ribulose diphosphate of 0.07 mM and a Km for HCO3- of 10 mM. Moreover, 6-phospho-D-gluconate was found to markedly inhibit the E. halophila carboxylase; a Ki for phosphogluconate of 0.14 mM was determined.     

Interaction of constituent subunits in ribulose 1,5-bisphosphate carboxylase from Aphanothece halophytica

Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) from the halophilic cyanobacterium, Aphanothece halophytica, dissociates into catalytic core (large subunit A oligomer) and small subunit B under low ionic strength during sucrose density gradient centrifugation. Supplementation of KCl, NaCl, or K2SO4 ( [I] = 0.3 M) partly prevents the dissociation, the preventive effect of divalent cation salts such as MgCl2 and CaCl2 being more effective than monovalent cation salts. RuBisCO with its higher-plant-type molecular form can be isolated from the cyanobacterial extracts using gradient medium containing 0.3 M KCl, 20 mM MgCl2, and 10 mM CaCl2. The isolated enzyme contains large subunit A and small subunit B in a molar ratio of approximately 1:1, estimated from the densitometric scanning of Coomassie blue-stained gels. During the second sucrose density gradient centrifugation to remove minor contaminants, a small amount of subunit B is depleted from the holoenzyme. Determination of the molecular weight by equilibrium centrifugation and electron microscopic observation have confirmed that the cyanobacterial RuBisCO has an A8B8-type structure. The enzyme activity per se is found to be sensitive to concentrations of salts, and small subunit B is obligatory for the enzyme catalysis. It has been shown that the more the enzyme activity is inhibited by salts, the tighter the association of subunit B becomes. It is likely that the active enzyme retains the loose conformational structure to such an extent that the dissociable release of subunit B from the holoenzyme in vivo is not allowed.     

Site-directed mutagenesis of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans

Using oligonucleotide-directed mutagenesis of the gene encoding the small subunit (rbcS) from Anacystis nidulans mutant enzymes have been generated with either Trp-54 of the small subunit replaced by a Phe residue, or with Trp-57 replaced by a Phe residue, whereas both Trp-54 and Trp-57 have been replaced by Phe residues in a double mutant. Trp-54 and Trp-57 are conserved in all amino acid sequences or the small subunit (S) that are known at present. The wild-type and mutant forms of Rubisco have all been purified to homogeneity. The wild-type enzyme, purified from Escherichia coli is indistinguishable from enzyme similarly purified from A. nidulans in subunit composition, subunit molecular mass and kinetic parameters (Vmax CO2 = 2.9 U/mg, Km CO2 = 155 microM). The single Trp mutants are indistinguishable from the wild-type enzyme by criteria (a) and (b). However, whereas, Km CO2 is also unchanged, Vmax CO2 is 2.5-fold smaller than the value for the wild-type enzyme for both mutants, demonstrating for the first time that single amino acid replacements in the non-catalytic small subunit influence the catalytic rate of the enzyme. The specificity factor tau, which measures the partitioning of the active site between the carboxylase and oxygenase reactions, was found to be invariant. Since tau is not affected by these mutations we conclude that S is an activating not a regulating subunit.     

A kinetic study of ribulose bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum.

The activation kinetics of purified Rhodospirillum rubrum ribulose bisphosphate carboxylase were analysed. The equilibrium constant for activation by CO(2) was 600 micron and that for activation by Mg2+ was 90 micron, and the second-order activation constant for the reaction of CO(2) with inactive enzyme (k+1) was 0.25 X 10(-3)min-1 . micron-1. The latter value was considerably lower than the k+1 for higher-plant enzyme (7 X 10(-3)-10 X 10(-3)min-1 . micron-1). 6-Phosphogluconate had little effect on the active enzyme, and increased the extent of activation of inactive enzyme. Ribulose bisphosphate also increased the extent of activation and did not inhibit the rate of activation. This effect might have been mediated through a reaction product, 2-phosphoglycolic acid, which also stimulated the extent of activation of the enzyme. The active enzyme had a Km (CO2) of 300 micron-CO2, a Km (ribulose bisphosphate) of 11--18 micron-ribulose bisphosphate and a Vmax. of up to 3 mumol/min per mg of protein. These data are discussed in relation to the proposed model for activation and catalysis of ribulose bisphosphate carboxylase.     

Catalytic properties of a hybrid between cyanobacterial large subunits and higher plant small subunits of ribulose bisphosphate carboxylase-oxygenase

The small subunits of spinach ribulosebisphosphate carboxylase-oxygenase were isolated by mild acid precipitation of the hexadecameric holoenzyme. About one-third of the small subunits remained in the supernatant while the remainder, and all of the large subunits, were precipitated and irreversibly denatured. The spinach small subunits were able to reassemble with the large subunit octamer of ribulosebisphosphate carboxylase-oxygenase from the cyanobacterium, Synechococcus ACMM 323, prepared as described previously (Andrews, T. J., and Ballment, B. (1983) J. Biol. Chem. 258, 7514-7518) to produce a catalytically active, hybrid enzyme. The heterologous small subunits bound an order of magnitude less tightly than homologous small subunits and the specific activity of the hybrid, when fully saturated with foreign small subunits, was about half that of the homologously reassembled or native Synechococcus enzyme. In addition, the Km(CO2) of the hybrid was about twice as high. However, the degree of partitioning between carboxylation and oxygenation was identical for the hybrid, the homologously reassembled, and the native Synechococcus enzymes and clearly less in favor of carboxylation than partitioning by the spinach enzyme. Therefore, this important facet of catalysis by ribulosebisphosphate carboxylase-oxygenase appears to be specified exclusively by the large subunit.     

Relationship between the Nucleo-Cytoplasm Composition and the Kinetic Properties of Ribulose-l,5-bisphosphate Carboxylase/Oxygenase of Rice

Ribulose-l,5-bisphosphate carboxylase/oxygenase (RubisCO, EC 4.1.1.39) was purified from leaves of rice (Oryza sativa), including four fertile cultivars and six male-sterile nuclear substitution lines: genome of cultivars in O. sativa L. f. spontanea cytoplasm and in O. sativa subsp. indica cv. Gambiaka Kokum cytoplasm, respectively. These RubisCO enzymes were divided into, two categories: (a) RubisCO with identical large subunits but different small subuinits, (b) RubisCO with variable large subunits and identical small subunits. Specific activities for both the carboxylation and oxygenation reactions of RubisCO were determined under standard conditions of activation. It was found that the Vmax(CO2) remained constant, but Km(O2) changed greatly, ranging from 223 μmol/L to 371 μmol/L in (a) group. The small subunits had significant effect on Km(O2) and on Vmax(O2)/Km(O2). No significant variation in Km(CO2), Vmax(O2), and the specificky factor were detected among the RubisCO with varied small subunits. Significant variation in Km(O2) and specificity factor were detected among the enzymes with varied large subunits, which also showed an important effect on Km(CO2) and Vmax(O2). RubisCO with heterologous large subunits had higher carboxylase activity and specificity factor than those with homologous large subunits.     

Properties of subcloned subunits of bacterial acetohydroxy acid synthases.

The acetohydroxy acid synthase (AHAS) isozymes from enterobacteria are each composed of a large and small subunit in an alpha 2 beta 2 structure. It has been generally accepted that the large (ca. 60-kDa) subunits are catalytic, while the small ones are regulatory. In order to further characterize the roles of the subunits as well as the nature and the specificities of their interactions, we have constructed plasmids encoding the large or small subunits of isozymes AHAS I and AHAS III, each with limited remnants of the other peptide. The catalytic properties of the large subunits have been characterized and compared with those of extracts containing the intact enzyme or of purified enzymes. Antisera to the isolated subunits have been used in Western blot (immunoblot) analyses for qualitative and semiquantitative determinations of the presence of the polypeptides in extracts. The large subunits of AHAS isozymes I and III have lower activities than the intact enzymes: Vmax/Km is 20 to 50 times lower in both cases. However, for AHAS I, most of this difference is due to the raised Km of the large subunit alone, while for AHAS III, it is due to a lowered Vmax. The substrate specificities, R, of large subunits are close to those of the intact enzymes. The catalytic activity of the large subunits of AHAS I is dependent on flavin adenine dinucleotide (FAD), as is that of the intact enzyme, although the apparent affinities of the large subunits alone for FAD are 10-fold lower. Isolated subunits are insensitive to valine inhibition. Nearly all of the properties of the intact AHAS isozyme I or III can be reconstituted by mixing extracts containing the respective large and small subunits. The mixing of subunits from different enzymes does not lead to activation of the large subunits. It is concluded that the catalytic machinery of these AHAS isozymes is entirely contained within the large subunits. The small subunits are required, however, for specific stabilization of an active conformation of the large subunits as well as for value sensitivity.     

Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions.

The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S. Knight, I. Andersson, and C.-I. Brändén [1990] J Mol Biol 215: 113-160). The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme. Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of an additional single residue (i.e. removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold. Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme. None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme. However, this truncated small subunit was still synthesized by the E. coli cells. These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme.     

Cloning of the alpha subunit of prolyl 4-hydroxylase from Drosophila and expression and characterization of the corresponding enzyme tetramer with some unique properties

Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha2beta2 tetramers, whereas the Caenorhabditis elegans enzyme is an alphabeta dimer, the beta subunit being identical to protein-disulfide isomerase (PDI). We report here that the processed Drosophila melanogaster alpha subunit is 516 amino acid residues in length and shows 34 and 35% sequence identities to the two types of human alpha subunit and 31% identity to the C. elegans alpha subunit. Its coexpression in insect cells with the Drosophila PDI polypeptide produced an active enzyme tetramer, and small amounts of a hybrid tetramer were also obtained upon coexpression with human PDI. Four of the five recently identified critical residues at the catalytic site were conserved, but a histidine that probably helps the binding of 2-oxoglutarate to the Fe2+ and its decarboxylation was replaced by arginine 490. The enzyme had a higher Km for 2-oxoglutarate, a lower reaction velocity, and a higher percentage of uncoupled decarboxylation than the human enzymes. The mutation R490H reduced the percentage of uncoupled decarboxylation, whereas R490S increased the Km for 2-oxoglutarate, reduced the reaction velocity, and increased the percentage of uncoupled decarboxylation. The recently identified peptide-binding domain showed a relatively low identity to those from other species, and the Km of the Drosophila enzyme for (Pro-Pro-Gly)10 was higher than that of any other animal prolyl 4-hydroxylase studied. A 1. 9-kilobase mRNA coding for this alpha subunit was present in Drosophila larvae.     

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.     

The large subunit determines catalytic specificity of barley sucrose:fructan 6-fructosyltransferase and fescue sucrose:sucrose 1-fructosyltransferase

Plant fructosyltransferases are highly homologous in primary sequence and typically consist of two subunits but catalyze widely different reactions. Using functional expression in the yeast Pichia pastoris, we show that the substrate specificity of festuca sucrose:sucrose 1--beta-D-fructosyltransferase (1-SST) and barley sucrose:fructan 6--beta-D-fructosyltransferase (6-SFT) is entirely determined by the large subunit. Chimeric enzymes with the large subunit of festuca 1-SST (LSuB) and the small subunit of barley 6-SFT have the same catalytic specificity as the native festuca 1-SST and vice versa. If the LSuB is expressed alone, it does not yield a functionally active enzyme, indicating that the small subunit is nevertheless essential.     

Evaluation by mutagenesis of the importance of 3 arginines in alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase

Mammalian NAD-dependent isocitrate dehydrogenase is an allosteric enzyme, activated by ADP and composed of 3 distinct subunits in the ratio 2alpha:1beta:1gamma. Based on the crystal structure of NADP-dependent isocitrate dehydrogenases from Escherichia coli, Bacillus subtilis, and pig heart, and a comparison of their amino acid sequences, alpha-Arg88, beta-Arg99, and gamma-Arg97 of human NAD-dependent isocitrate dehydrogenase were chosen as candidates for mutagenesis to test their roles in catalytic activity and ADP activation. A plasmid harboring cDNA that encodes alpha, beta, and gamma subunits of the human isocitrate dehydrogenase (Kim, Y. O., Koh, H. J., Kim, S. H., Jo, S. H., Huh, J. W., Jeong, K. S., Lee, I. J., Song, B. J., and Huh, T. L. (1999) J. Biol. Chem. 274, 36866-36875) was used to express the enzyme in isocitrate dehydrogenase-deficient E. coli. Wild type (WT) and mutant enzymes (each containing 2 normal subunits plus a mutant subunit with alpha-R88Q, beta-R99Q, or gamma-R97Q) were purified to homogeneity yielding enzymes with 2alpha:1beta:1gamma subunit composition and a native molecular mass of 315 kDa. Specific activities of 22, 14, and 2 micromol of NADH/min/mg were measured, respectively, for WT, beta-R99Q, and gamma-R97Q enzymes. In contrast, mutant enzymes with normal beta and gamma subunits and alpha-R88Q mutant subunit has no detectable activity, demonstrating that, although beta-Arg99 and gamma-Arg97 contribute to activity, alpha-Arg88 is essential for catalysis. For WT enzyme, the Km for isocitrate is 2.2 mm, decreasing to 0.3 mm with added ADP. In contrast, for beta-R99Q and gamma-R97Q enzymes, the Km for isocitrate is the same in the absence or presence of ADP, although all the enzymes bind ADP. These results suggest that beta-Arg99 and gamma-Arg97 are needed for normal ADP activation. In addition, the gamma-R97Q enzyme has a Km for NAD 10 times that of WT enzyme. This study indicates that a normal alpha subunit is required for catalytic activity and alpha-Arg88 likely participates in the isocitrate site, whereas the beta and gamma subunits have roles in the nucleotide functions of this allosteric enzyme.     

Assessment of structural and functional divergence far from the large subunit active site of ribulose-1,5-bisphosphate carboxylase/oxygenase

Despite conservation of three-dimensional structure and active-site residues, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) enzymes from divergent species differ with respect to catalytic efficiency and CO2/O2 specificity. A deeper understanding of the structural basis for these differences may provide a rationale for engineering an improved enzyme, thereby leading to an increase in photosynthetic CO2 fixation and agricultural productivity. By comparing 500 active-site large subunit sequences from flowering plants with that of the green alga Chlamydomonas reinhardtii, a small number of residues were found to differ in regions previously shown by mutant screening to influence CO2/O2 specificity. When directed mutagenesis and chloroplast transformation were used to change Chlamydomonas Met-42 and Cys-53 to land plant Val-42 and Ala-53 in the large subunit N-terminal domain, little or no change in Rubisco catalytic properties was observed. However, changing Chlamydomonas methyl-Cys-256, Lys-258, and Ile-265 to land plant Phe-256, Arg-258, and Val-265 at the bottom of the alpha/beta-barrel active site caused a 10% decrease in CO2/O2 specificity, largely due to an 85% decrease in carboxylation catalytic efficiency (Vmax/Km). Because land plant Rubisco enzymes have greater CO2/O2 specificity than the Chlamydomonas enzyme, this group of residues must be complemented by other residues that differ between Chlamydomonas and land plants. The Rubisco x-ray crystal structures indicate that these residues may reside in a variable loop of the nuclear-encoded small subunit, more than 20 A away from the active site.     

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.     

Activation and regulation of ribulose bisphosphate carboxylase-oxygenase in the absence of small subunits.

Ribulose 1,5-bisphosphate carboxylase from Rhodospirillum rubrum requires CO2 and Mg2+ for activation of both CO2, both the carboxylase and oxygenase activities are stimulated by 6-phoshpo-D-gluconate, fructose 1,6-bisphosphate, 2-phosphoglycolate, 3-phosphoglycerate, NADPH, and fructose 6-phosphate. The carboxylase activity is not activated by ribose 5-phosphate. The substrate, ribulose bisphosphate, neither activates nor inhibits the CO2 and Mg2+ activation of this enzyme. Activation by CO2 and Mg2+ is rapid and results in increased susceptibility to active-site-directed protein modification reagents. Because the R. rubrum carboxylase-oxygenase is a dimer of large subunits and contains no small subunits, these results suggest that the effector binding sites of the higher plant enzyme may also be found on the large subunit.