Modulation of the tight binding of carboxyarabinitol 1,5-bisphosphate to the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase

The large subunit (L) of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) from Synechococcus PCC 6301 was expressed in Escherichia coli, purified as the octamer L8, and analyzed for its ability to tightly bind the transition state analog, 2-carboxyarabinitol 1,5-bisphosphate (CABP). [14C]CABP remained tightly bound to L8 after challenging with [12C]CABP and gel filtration, indicating that L8 alone without the small subunit (S) could tightly bind CABP. Binding of CABP to L8 induced a shift in the gel filtration profile due to apparent aggregation of L8. Aggregation did not occur with the L8S8-CABP complex nor with L8-CABP in the presence of 150 mM MgCl2. If ionic strength was increased with either KCl or MgCl2 during or after the binding of [14C]CABP to L8, [14C]CABP in the complex exchanged with [12C]CABP and was lost from the protein. Ionic strength strongly affected the rate constant (k4) for [14C]CABP dissociation from the L8-[14C]CABP complex, but had little effect on k4 for the L8S8-CABP complex. The differences in CABP binding characteristics between the L8-CABP and L8S8-CABP complexes demonstrate that S is intimately involved in maintaining the stability of the tight binding of CABP to the active site. These are the same interactions stabilizing the intermediate, 3-keto-2-carboxyarabinitol 1,5-bisphosphate, to native rubisco during CO2 fixation.     

The relative catalytic specificities of the large subunit core of Synechococcus ribulose bisphosphate carboxylase/oxygenase

The relative specificities of the carboxylase and oxygenase reactions catalyzed by the recombinant large subunit core (L8) of Synechococcus ribulose 1,5-bisphosphate carboxylase have been determined. The L8 core still retained the ability to catalyze both reactions but at a much reduced turnover rate, about 0.6% of the holoenzyme. The fate of ribulose 1,5-bisphosphate during carboxylation and oxygenation by L8 was compared with the Synechococcus holoenzyme (reconstituted from L8 and recombinant small subunits), the carboxylase from Rhodospirullum rubrum, and that of spinach. The absence of small subunits had no significant effect on the partitioning of the bisphosphate substrate between the two reactions. Thus the course of the two competing reactions is a characteristic of the structural elements that compose the L-subunits, whereas the S-subunits exert their effect on factors common to both reactions such as the specificity of the bisphosphate substrate.     

Linked Rubisco subunits can assemble into functional oligomers without impeding catalytic performance

Although transgenic manipulation in higher plants of the catalytic large subunit (L) of the photosynthetic CO2-fixing enzyme ribulose 1,5-bisphospahte carboxylase/oxygenase (Rubisco) is now possible, the manipulation of its cognate small subunit (S) is frustrated by the nuclear location of its multiple gene copies. To examine whether L and S can be engineered simultaneously by fusing them together, the subunits from Synechococcus PCC6301 Rubisco were tethered together by different linker sequences, producing variant fusion peptides. In Escherichia coli the variant PCC6301 LS fusions assembled into catalytically functional octameric ([LS]8) and hexadecameric ([[LS]8]2) quaternary structures that excluded the integration of co-expressed unfused S. Assembly of the LS fusions into Rubisco complexes was impaired 50-90% relative to the assembly of unlinked L and S into L8S8 enzyme. Assembly in E. coli was not emulated using tobacco SL fusions that accumulated entirely as insoluble protein. Catalytic measurements showed the CO2/O2 specificity, carboxylation rate, and Michaelis constants for CO2 and ribulose 1,5-bisphosphate for the cyanobacterial Rubisco complexes comprising fusions where the S was linked to the N terminus of L closely matched those of the wild-type L8S8 enzyme. In contrast, the substrate affinities and carboxylation rate of the Rubisco complexes comprising fusions where L was fused to the N terminus of S or a six-histidine tag was appended to the C terminus of L were compromised. Overall this work provides a framework for implementing an alternative strategy for exploring simultaneous engineering of modified, or foreign, Rubisco L and S subunits in higher plant plastids.     

Status of the substrate binding sites of ribulose bisphosphate carboxylase as determined with 2-C-carboxyarabinitol 1,5-bisphosphate

The properties of the tight and specific binding of 2-C-carboxy-d-arabinitol 1,5-bisphosphate (CABP), which occurs only to reaction sites of ribulose 1,5-bisphosphate carboxylase (Rubisco) that are activated by CO₂ and Mg²⁺, were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of [¹⁴C]CABP fit a multiple exponential rate equation with half of the sites binding with a rate constant of 40 per minute and the second half of the sites binding at 3.2 per minute. This suggests that after CABP binds to one site of a dimer of Rubisco large subunits, binding to the second site is considerably slower, indicating negative cooperativity as previously reported (S Johal, BE Partridge, R Chollet [1985] J Biol Chem 260: 9894-9904). The rate of CABP binding to partially activated Rubisco was complete within 2 to 5 minutes, with slower binding to inactive sites as they formed the carbamate and bound Mg²⁺. Addition of [¹⁴C]CABP and EDTA stopped binding of Mg²⁺ and allowed tight binding of the radiolabel only to sites which were CO₂/Mg²⁺-activated at that moment. This approach estimated the amount of CO₂/Mg²⁺-activated sites in the presence of inactive sites and carbamylated sites lacking Mg²⁺. The rate of CO₂ fixation was proportional to the CO₂/Mg²⁺-activated sites. During light-dependent CO₂ fixation with isolated spinach chloroplasts, the amount of carbamylation was proportional to Rubisco activity either initially upon lysis of the plastids or following total activation with Mg²⁺ and CO₂. Lysis of chloroplasts in media with [¹⁴C]CABP plus EDTA estimated those carbamylated sites having Mg²⁺. The loss of Rubisco activation during illumination was partially due to the lack of Mg²⁺ to stabilize the carbamylated sites.     

Mutations in the small subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase increase the formation of the misfire product xylulose-1,5-bisphosphate.

The small subunit (S) increases the catalytic efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) by stabilizing the active sites generated by four large subunit (L) dimers. This stabilization appears to be due to an influence of S on the reaction intermediate 2,3-enediol, which is formed after the abstraction of a proton from the substrate ribulose-1,5-bisphosphate. We tested the functional significance of residues that are conserved among most species in the carboxy-terminal part of S and analyzed their influence on the kinetic parameters of Synechococcus holoenzymes. The replacements in S (F92S, Q99G, and P108L) resulted in catalytic activities ranging from 95 to 43% of wild type. The specificity factors for the three mutant enzymes were little affected (90-96% of wild type), but Km(CO2) values increased 0.5- to 2-fold. Mutant enzymes with replacements Q99G and P108L showed increased mis-protonation, relative to carboxylation, of the 2,3-enediol intermediate, forming 2 to 3 times more xylulose-1,5-bisphosphate per ribulose-1,5-bisphosphate utilized than wild-type or F92S enzymes. The results suggest that specific alterations of the L/S interfaces and of the hydrophobic core of S are transmitted to the active site by long-range interactions. S interactions with L may restrict the flexibility of active-site residues in L.     

Artificially evolved Synechococcus PCC6301 Rubisco variants exhibit improvements in folding and catalytic efficiency

The photosynthetic CO2-fixing enzyme, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), is responsible for most of the world's biomass, but is a slow non-specific catalyst. We seek to identify and overcome the chemical and biological constraints that limit the evolutionary potential of Rubisco in Nature. Recently, the horizontal transfer of Calvin cycle genes (rbcL, rbcS and prkA) from cyanobacteria (Synechococcus PCC6301) to gamma-proteobacteria (Escherichia coli) was emulated in the laboratory. Three unique Rubisco variants containing single (M259T) and double (M259T/A8S, M259T/F342S) amino acid substitutions in the L (large) subunit were identified after three rounds of random mutagenesis and selection in E. coli. Here we show that the M259T mutation did not increase steady-state levels of rbcL mRNA or L protein. It instead improved the yield of properly folded L subunit in E. coli 4-9-fold by decreasing its natural propensity to misfold in vivo and/or by enhancing its interaction with the GroES-GroEL chaperonins. The addition of osmolites to the growth media enhanced productive folding of the M259T L subunit relative to the wild-type L subunit, while overexpression of the trigger factor and DnaK/DnaJ/GrpE chaperones impeded Rubisco assembly. The evolved enzymes showed improvement in their kinetic properties with the M259T variant showing a 12% increase in carboxylation turnover rate (k(c)cat), a 15% improvement in its K(M) for CO2 and no change in its K(M) for ribulose-1,5-bisphosphate or its CO2/O2 selectivity. The results of the present study show that the directed evolution of the Synechococcus Rubisco in E. coli can elicit improvements in folding and catalytic efficiency.     

A Mutant Isolated from the Cyanobacterium Synechococcus PCC7942 Is Unable to Adapt to Low Inorganic Carbon Conditions

Using a novel screening procedure, we have selected a new class of mutant from the cyanobacterium Synechococcus PCC7942 that fails to adapt to growth at an extremely low inorganic carbon (Ci) concentration. The mutant (Tm17) reported in this study grows normally at or above air levels of CO2 (340 [mu]L L-1) but does not survive at 20 [mu]L L-1 CO2 in air. Air-grown Tm17 cells showed properties similar to wild-type cells in various aspects of the CO2-concentrating mechanism examined. Following transfer from air levels to 20 [mu]L L-1 CO2, however, the mutant cells failed to increase their photosynthetic affinity for Ci. This results in an approximately 10-fold difference in photosynthetic affinity between the wild-type and Tm17 cells under Ci-limiting conditions [the K0.5(Ci) values were 11 and 136 [mu]M, respectively]. Further examination of factors possibly contributing to this low photosynthetic affinity showed that Tm17 cells have no inducible high-affinity HCO3- transport and do not appear to show induction of increased carboxysomal carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase activities. It appears that a common factor, possibly relating to CO2 detection and/or induction signal, or the HCO3-transport mechanism may have been impaired in the mutant. Complementation results indicate that the mutation responsible for the phenotype has occurred in an 8- to 10-kb EcoRI genomic DNA fragment.     

Crystal structure of rice Rubisco and implications for activation induced by positive effectors NADPH and 6-phosphogluconate

The key enzyme of plant photosynthesis, D-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), must be activated to become catalytically competent via the carbamylation of Lys201 of the large subunit and subsequent stabilization by Mg(2+) coordination. Many biochemical studies have reported that reduced nicotinamide adenine dinucleotide phosphate (NADPH) and 6-phosphogluconate (6PG) function as positive effectors to promote activation. However, the structural mechanism remains unknown. Here, we have determined the crystal structures of activated rice Rubisco in complex with NADPH, 6PG, or 2-carboxy-D-arabinitol 1,5-bisphosphate (2CABP). The structures of the NADPH and 6PG complexes adopt open-state conformations, in which loop 6 at the catalytic site and some other loops are disordered. The structure of the 2CABP complex is in a closed state, similar to the previous 2CABP-bound activated structures from other sources. The catalytic sites of the NADPH and 6PG complexes are fully activated, despite the fact that bicarbonate (NaHCO(3)) was not added into the crystallization solution. In the catalytic site, NADPH does not interact with Mg(2+) directly but interacts with Mg(2+)-coordinated water molecules, while 6PG interacts with Mg(2+) directly. These observations suggest that the two effectors promote Rubisco activation by stabilizing the complex of Mg(2+) and the carbamylated Lys201 with unique interactions and preventing its dissociation. The structure also reveals that the relaxed complex of the effectors (NADPH or 6PG), distinct from the tight-binding mode of 2CABP, would allow rapid exchange of the effectors in the catalytic sites by substrate D-ribulose 1,5-bisphosphate for catalysis in physiological conditions.     

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.     

Comparative affinities of the epimeric reaction-intermediate analogs 2- and 4-carboxy-D-arabinitol 1,5-bisphosphate for spinach ribulose 1,5-bisphosphate carboxylase

2-Carboxy-3-keto-D-arabinitol 1,5-bisphosphate is a tightly bound intermediate of the carboxylase reaction of ribulosebisphosphate carboxylase/oxygenase. Two stereoisomers of an analog of this intermediate, 2-carboxy-D-arabinitol 1,5-bisphosphate (2CABP) and 4-carboxy-D-arabinitol 1,5-bisphosphate (4CABP), are exceptionally potent, virtually irreversible inhibitors of the spinach carboxylase, presumably due to their structural similarity to the gem-diol (hydrated carbonyl at C-3) form of the intermediate. Incubation of the enzyme with either leads to time-dependent loss of activity. Inhibition of the enzyme is biphasic, with initial dissociation constants of 0.47 and 0.19 microM and maximal rates for tight complex formation of 2.2 and 1.8 min-1 for 2CABP and 4CABP, respectively. These values give second-order rate constants for tight complex formation of 7.8 x 10(4) and 1.6 x 10(5) M-1 s-1. To determine the overall affinity of the spinach enzyme for 2CABP and 4CABP, the release rates were determined by dual isotope exchange (3H-inhibitor complex with free 14C-inhibitor). Exchange half-times of 1.82 and 530 days were observed for 4CABP and 2CABP, respectively. Overall dissociation constants of 28 pM (2.8 x 10(-11) M) and 190 fM (1.9 x 10(-13) M) were calculated from these dissociation rates together with the rates of association determined by inactivation kinetics. The difference in affinity of 2CABP and 4CABP corresponds to 2.9 kcal/mol, presumably reflecting the difference in interaction of the enzyme with the two hydroxyls of the intermediate's gem-diol. The kinetic behavior of these two inhibitors, in particular the rather slow maximal rates of association, are consistent with the expected behavior of analogs of a labile intermediate of an enzymic reaction that is far more stable than a transition state.     

The role of chloroplast electron transport and metabolites in modulating Rubisco activity in tobacco. Insights from transgenic plants with reduced amounts of cytochrome b/f complex or glyceraldehyde 3-phosphate dehydrogenase

Leaf metabolites, adenylates, and Rubisco activation were studied in two transgenic tobacco (Nicotiana tabacum L. cv W38) types. Plants with reduced amounts of cytochrome b/f complex (anti-b/f) have impaired electron transport and a low transthylakoid pH gradient that restrict ATP and NADPH synthesis. Plants with reduced glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) have a decreased capacity to use ATP and NADPH in carbon assimilation. The activation of the chloroplast NADP-malate dehydrogenase decreased in anti-b/f plants, indicating a low NADPH/NADP(+) ratio. The whole-leaf ATP/ADP in anti-b/f plants was similar to wild type, while it increased in anti-GAPDH plants. In both plant types, the CO(2) assimilation rates decreased with decreasing ribulose 1, 5-bisphosphate concentrations. In anti-b/f plants, CO(2) assimilation was further compromised by reduced carbamylation of Rubisco, whereas in anti-GAPDH plants the carbamylation remained high even at subsaturating ribulose 1,5-bisphosphate concentrations. We propose that the low carbamylation in anti-b/f plants is due to reduced activity of Rubisco activase. The results suggest that light modulation of activase is not directly mediated via the electron transport rate or stromal ATP/ADP, but some other manifestation of the balance between electron transport and the consumption of its products. Possibilities include the transthylakoid pH gradient and the reduction state of the acceptor side of photosystem I and/or the degree of reduction of the thioredoxin pathway.     

In situ assay of ribulose-1,5-bisphosphate carboxylase/oxygenase in Thiobacillus neapolitanus.

Cells permeabilized with chloroform yielded ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activities nearly equal to those of cell extracts, thus indicating that both cytoplasmic and carboxysomal RuBisCO are functional in situ. The carboxysomal and cytoplasmic RuBisCO both form the CO2-Mg2(+)-enzyme ternary complex, as evidenced by stabilization with 2-C-carboxy-D-arabinitol-1,5-bisphosphate (CABP), a potent competitive inhibitor of RuBisCO. The data are consistent with the hypothesis that the carboxysome is functional in carbon dioxide fixation.     

A crystal form of ribulose-1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum in the activated state

A new crystal form of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from Nicotiana tabacum has been obtained at alkaline pH with polyethylene glycol 8000 in the presence of a non-ionic detergent, beta-octyl glucoside. The crystals are grown at room temperature by the hanging-drop vapor diffusion technique from a protein solution containing enzyme complexed with CO2, Mg2+, and the transition state analog 2-C-carboxy-D-arabinitol-1,5-bisphosphate. The crystals belong to the the space group P3(1)21 (or P3(2)21) with the cell parameters a = 204.6 A, and c = 117.4 A (1 A = 0.1 nm). The asymmetric unit contains half (L4S4: L, large subunit, 53,000 Mr; S, small subunit, 15,000 Mr) of a hexadecameric molecule (L8S8, 540,000 Mr). The crystals diffract to at least 2.6 A Bragg spacing and are suitable for X-ray structure determination.     

A spectrometric study of the Co(2+)-activated ribulose-1,5-bisphosphate carboxylase reaction during turnover and at different pH.

EPR as well as optical absorption studies of the Co(2+)-activated ribulose-1,5-bisphosphate carboxylase/oxygenase under turnover conditions show that the formation of the two detectable intermediates are pH dependent. The amount of one of them, which earlier has been proposed to be a metal coordinated endiol of ribulose-1,5-bisphosphate (Br?ndén et al. (1987) Biochim. Biophys. Acta 916, 298-303), increased with increasing pH. Distinct optical absorption bands could be assigned to this intermediate and a pH profile could be made. It is therefore proposed that a base with a pKa value of about 8 is responsible for the enzyme-catalysed abstraction of a proton from ribulose-1,5-bisphosphate in order to form the metal coordinated endiol of ribulose-1,5-bisphosphate.     

Calcium supports loop closure but not catalysis in Rubisco

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses CO(2) assimilation in biology. A prerequisite for catalysis is an activation process, whereby an active site lysine is selectively carbamylated. The carbamyl group is then stablised by a metal ion, which in vivo is Mg(2+). Other divalent metal ions can replace Mg(2+) as activators in vitro, but the nature of the metal ion strongly influences the catalytic activity of the enzyme and has a differential effect on the ratio of the carboxylation reaction and the competing oxygenation reaction. Biochemical studies show that calcium promotes carbamylation but not catalysis. To investigate the role of the metal in catalysis, we have determined two structures of the enzyme complexed with Ca(2+) and the transition state analogue 2-carboxy-D-arbinitol-1,5-bisphosphate (2CABP). One of the complexes was prepared by soaking 2CABP into crystals of the enzyme-Ca(2+)-product complex, while the other was obtained by cocrystallising the enzyme with calcium and 2CABP under activating conditions. The two crystals belong to different space groups, and one was merohedrally twinned. Both complexes show very similar three-dimensional features. The enzyme is carbamylated at Lys201, and requisite loops close over the bound ligands in the active site, shielding them from the solvent in a manner similar to the corresponding complex with Mg(2+). However, there are subtle differences that could explain the particular role of Ca(2+) in these processes. The larger radius of the calcium ion and its reduced Lewis-acid character causes a significant increase in the required proton hop distance between the C3 proton and the carbamate on Lys201 in the calcium complex. This alone could explain the inability of calcium to sustain catalysis in Rubisco. Similar effects are also expected on subsequent proton transfer steps in the catalytic cycle. Here we also discuss the effect of metal substitution on the dynamics of the ligands around the metal ion.     

Subsaturating Ribulose-1,5-Bisphosphate Concentration Promotes Inactivation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) (Studies Using Continuous Substrate Addition in the Presence and Absence of Rubisco Activase)

We developed a continuous-addition method for maintaining subsaturating concentrations of ribulose-1,5-bisphosphate (RuBP) for several minutes, while simultaneously monitoring its consumption by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This method enabled us to observe the effects of subsaturating RuBP and CO2 concentrations on the activity of Rubisco during much longer periods than previously studied. At saturating CO2, the activity of the enzyme declined faster when RuBP was maintained at concentrations near its Km value than when RuBP was saturating. At saturating RuBP, activity declined faster at limiting than at saturating CO2, in accordance with previous observations. The most rapid decline in activity occurred when both CO2 and RuBP concentrations were subsaturating. The activity loss was accompanied by decarbamylation of the enzyme, even though the enzyme was maintained at the same CO2 concentration before and after exposure to RuBP. Rubisco activase ameliorated the decline in activity at subsaturating CO2 and RuBP concentrations. The results are consistent with a proposed mechanism for regulating the carbamylation of Rubisco, which postulates that Rubisco activase counteracts Rubisco's unfavorable carbamylation equilibrium in the presence of RuBP by accelerating, in an ATP-dependent manner, the release of RuBP from its complex with uncarbamylated sites.     

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.     

Catalytic properties of recombinant octameric, hexadecameric, and heterologous cyanobacterial/bacterial ribulose- 1,5-bisphosphate carboxylase/oxygenase

The recent isolation of a catalytically competent recombinant octameric core of the hexadecameric ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans (Synechococcus) (B. Lee and F. R. Tabita, 1990, Biochemistry 29, 9352-9357) has provided a useful system for examining the properties of this enzyme in the absence of small subunits. Unlike most sources of hexadecameric ribulose bisphosphate carboxylase, the nonactivated Anacystis holoenzyme was not inhibited markedly by preincubation with ribulose 1,5-bisphosphate. This was also true for the Anacystis octameric core and a heterologous recombinant enzyme that comprised large subunits from Anacystis and small subunits from the bacterium Alcaligenes eutrophus, suggesting that substrate-mediated inactivation is not influenced by small subunits. In addition, the CO2/O2 specificity factor was not affected by the source of the small subunits incorporated into the structure of the hexadecameric protein, in agreement with previous in vitro heterologous reconstitution studies. The activated octameric Anacystis enzyme, however, was significantly more sensitive to inhibition by the phosphorylated effector 6-phosphogluconate than were the hexadecameric Alcaligenes and Anacystis enzymes and the heterologous Anacystis-Alcaligenes hybrid.     

Characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase carrying ribulose 1,5-bisphosphate at its regulatory sites and the mechanism of interaction of this form of the enzyme with ribulose-1,5-bisphosphate-carboxylase/oxygenase activase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu(1,5)P2CO] from plant sources shows a biphasic reaction course when assayed with more than 2 mM ribulose 1,5-bisphosphate [Rbu(1,5)P2]. In the burst, Rbu(1,5)P2CO has its substrate-binding sites occupied with Rbu(1,5)P2 for the initial few minutes, then both substrate-binding and regulatory sites are occupied by Rbu(1,5)P2 in the subsequent linear phase, at physiological concentrations of Rbu(1,5)P2 [A. Yokota (1991) J. Biochem. (Tokyo) 110, 246-252]. This study attempts the characterization of spinach Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the regulatory sites and the interaction of Rbu(1,5)P2CO activase with Rbu(1,5)P2CO purified with poly(ethylene glycol) 4000 without denaturation. Binding of Rbu(1,5)P2 to the regulatory sites strongly influences the temperature dependence of the carboxylase activity of Rbu(1,5)P2CO. The activation energy of Rbu(1,5)P2CO with Rbu(1,5)P2 at the regulatory sites was 40% larger than that without Rbu(1,5)P2 over 30 degrees C, although the binding did not affect the activation energy below this temperature. This caused the almost linear reaction course of the carboxylase reaction at 50 degrees C. The optimum pH for the activity of Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the sites was 8.0-8.2, and increased by about pH 0.2 from that of Rbu(1,5)P2CO without Rbu(1,5)P2. The ratio of the activity of the former form to that of the latter increased with increasing pH with an inflection point at pH 8.1. The increase in the ratio was accompanied by a decrease in the hysteric conformational change of Rbu(1,5)P2CO. The ATP-hydrolyzing activity inherent to Rbu(1,5)P2CO activase was stimulated about twofold by 3-5 mM Rbu(1,5)P2. Rbu(1,5)P2CO in the inactive complex with Rbu(1,5)P2 experienced hysteresis and bound Rbu(1,5)P2 at the regulatory sites during activation in the presence of Rbu(1,5)P2CO activase. Evidence was obtained that Rbu(1,5)P2CO activase promoted the activation of Rbu(1,5)P2CO through binding to the large subunits of Rbu(1,5)P2CO.     

Ribulose 1,5-bisphosphate carboxylase/oxygenase from Pseudomonas oxalacticus.

Ribulose 1,5-bisphosphate carboxylase/oxygenase was purified by a rapid, facile procedure from formate-grown Pseudomonas oxalaticus. The electrophoretically homogeneous enzyme had specific activities of 1.9 mumol of CO2 fixed per min per mg of protein and 0.15 mumol of O2 consumed per min per mg of protein. The amino acid composition was similar to that of other bacterial sources of the enzyme. The molecular weights determined by sedimentation equilibrium and by gel filtration were 421,000 and 450,000, respectively. Upon sodium dodecyl sulfate electrophoresis of enzyme purified under conditions which would limit proteolysis, two types of large (L) subunits and two types of small (S) subunits were observed with apparent molecular weights of 57,000, 55,000, 17,000 and 15,000. By densitometric scans at two different protein concentrations the stoichiometry of the total large to total small subunits was 1:1, implying an L6S6 structure. Electron micrographs of the enzyme revealed an unusual structure that was inconsistent with a cubical structure. The enzyme had an unusually high Km for ribulose 1,5-bisphosphate (220 microM) and was strongly inhibited by 6-phosphogluconate in the ribulose 1,5-bisphosphate carboxylase assay (Ki = 270 microM). One, 5, and 12 days after purification the enzyme was half-maximally activated at 0.13 microM, 0.23 mM, and 0.70 mM CO2, respectively, at saturating Mg2+. At saturating CO2, enzyme 1 day afer purification responded sigmoidally to Mg2+ and was half-maximally activated by 0.85 mM Mg2+ in the absence of 6-phosphogluconate (Hill coefficient, h = 2.0) and by 0.19 mM Mg2+ in the presence of mM 6-phosphogluconate (h = 1.7).