Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by ribulose-1,5-bisphosphate epimerization and degradation products

Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P₂) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P₂, is also present. Both compounds simulate the substrate inhibition of ribulose-P₂ carboxylase/oxygenase previously reported for ribulose-P₂. Freshly prepared ribulose-P₂ had little inhibitory activity. The instability of ribulose-P₂ may be one reason for a high level of ribulose-P₂ carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P₂. Because the K_D of the enzyme/substrate complex is ≤1 μM, all ribulose-P₂ generated in situ may be stored as this complex to prevent decomposition.     

Dissociation of ribulose-1,5-bisphosphate bound to ribulose-1,5-bisphosphate carboxylase/oxygenase and its enhancement by ribulose-1,5-bisphosphate carboxylase/oxygenase activase-mediated hydrolysis of ATP

Ribulose bisphosphate (RuBP), a substrate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is an inhibitor of Rubisco activation by carbamylation if bound to the inactive, noncarbamylated form of the enzyme. The effect of Rubisco activase on the dissociation kinetics of RuBP bound to this form of the enzyme was examined and characterized with the use of ³H-labeled RuBP and proteins purified from spinach (Spinacia oleracea L.) In the absence of Rubisco activase and in the presence of a large excess of unlabeled RuBP, the dissociation rate of bound [1-³H]RuBP was much faster after a short (30 second) incubation than after an extended incubation (1 hour). After 1 hour of incubation, the dissociation rate constant (K_off) of the bound RuBP was 4.8 × 10⁻⁴ per second, equal to a half-time of about 35 minutes, whereas the rate after only 30 seconds was too fast to be accurately measured. This time-dependent change in the dissociation rate was reflected in the subsequent activation kinetics of Rubisco in the presence of RuBP, CO₂, and Mg²⁺, and in both the absence or presence of Rubisco activase. However, the activation of Rubisco also proceeded relatively rapidly without Rubisco activase if the RuBP level decreased below the estimated catalytic site concentration. High pH (pH 8.5) and the presence of Mg²⁺ in the medium also enhanced the dissociation of the bound RuBP from Rubisco in the presence of RuBP. In the presence of Rubisco activase, Mg²⁺, ATP (but not the nonhydrolyzable analog, adenosine-5′-O-[3-thiotriphosphate]), excess RuBP, and an ATP-regenerating system, the dissociation of [1-³H]RuBP from Rubisco was increased in proportion to the amount of Rubisco activase added. This result indicates that Rubisco activase-mediated hydrolysis of ATP is required for promotion of the enhanced dissociation of the bound RuBP from Rubisco. Furthermore, product analysis by ion-exchange chromatography demonstrated that the release of the bound RuBP, in an unchanged form, was considerably faster than the observed increase in Rubisco activity. Thus, RuBP dissociation was experimentally separated from activation and precedes the subsequent formation of active, carbamylated Rubisco during activation of Rubisco by Rubisco activase.     

Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis decays steadily when exposed to agents that induce oxidative modification of cysteine residues (Cu²⁺, benzofuroxan, disulfides, arsenite, oxidized ascorbate). Inactivation takes place with a concomitant loss of cysteine sulfhydryl groups and dimerization of large subunits of the enzyme. 40% activity loss induced by the vicinal thiol-reagent arsenite is caused by modification of a few neighbor residues while the almost complete inactivation achieved with disulfides is due to extensive oxidation leading to formation of mixed disulfides with critical cysteines of the protein. In most cases oxidative inactivation is also accompanied by an increased sensitivity to proteolysis by trypsin, chymotrypsin or proteinase K. Both enzymatic activity and resistance to proteolysis can be restored through treatment with several thiols (cysteamine, cysteine, dithiothreitol and, more slowly, reduced glutathione). Redox effectors which are thought to regulate the chloroplast activity (NADPH, ferredoxin and thioredoxin) do not reactivate the oxidized enzyme. When ribulose-1,5-bisphoshate carboxylase/oxygenase is incubated with cystamine/cysteamine mixtures having different disulfide/thiol ratio (r), inactivation takes place around r=1.5 while proteolytic sensitization occurs under more oxidative conditions (r=4). It is suggested that oxidative modification may happen in vivo under exceptional circumstances, such as senescence, bleaching or different kinds of stress, leading to enzyme inactivation and triggering the selective degradation of the carboxylase that has been repeatedly observed during these processes.     

Dissociation of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach by urea

The dissociation of D-ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach, which consists of eight large subunits (L, 53 kDa) and eight small subunits (S, 14 kDa) and thus has a quarternary structure L8S8, has been investigated using a variety of physical techniques. Gel chromatography using Sephadex G-100 indicates the quantitative dissociation of the small subunit S from the complex at 3-4 M urea (50 mM Tris/Cl pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol and 5 mM 2-mercaptoethanol). The dissociated S is monomeric. Analytical ultracentrifuge studies show that the core of large subunits, L, remaining at 3-4 M urea sediments with S20, w = 15.0 S, whereas the intact enzyme (L8S8) sediments with S20, w = 17.7S. The observed value is consistent with a quarternary structure L8. The dissociation reaction in 3-4 M urea can thus be represented by L8S8----L8 + 8S. At urea concentrations c greater than 5 M the L8 core dissociates into monomeric, unfolded large subunits. A large decrease in fluorescence emission intensity accompanies the dissociation of the small subunit S. This change is completed at 4 M urea. No changes are observed upon dissociating the L8 core. The kinetics of dissociation of the small subunit, as monitored by fluorescence spectroscopy, closely follow the kinetics of loss of carboxylase activity of the enzyme. Studies of the circular dichroism of D-ribulose-1,5-bisphosphate carboxylase in the wavelength region 200-260 nm indicate two conformational transitions. The first one ([0]220 from -8000 to -3500 deg cm2 dmol-1) is completed at 4 M urea and corresponds to the dissociation of the small subunit and coupled conformational changes. The second one ([0]220 from -3500 to -1200 deg cm2 dmol-1) is completed at 6 M urea and reflects the dissociation and unfolding of large subunits from the core. The effect of activation of the enzyme by addition of MgCl2 (10 mM) and NaHCO3 (10 mM) on these conformational transitions was investigated. The first conformational transition is then shifted to higher urea concentrations: a single transition ([0]220 from -8000 to -1200 deg cm2 dmol-1) is observed for the activated enzyme. From the urea dissociation experiments we conclude that both large (L) and small (S) subunits are important for carboxylase activity of spinach D-ribulose-1,5-bisphosphate carboxylase: the L-S subunit interactions tighten upon activation and dissociation of S leads to a coupled, proportional loss of enzyme activity.     

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.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase protein prevents the in vitro decline in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase

The rate of CO₂ fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) following addition of ribulose 1,5-bisphosphate (RuBP) to fully activated enzyme, declined with first-order kinetics, resulting in 50% loss of rubisco activity after 10 to 12 minutes. This in vitro decline in rubisco activity, termed fall-over, was prevented if purified rubisco activase protein and ATP were added, allowing linear rates of CO₂ fixation for up to 20 minutes. Rubisco activase could also stimulate rubisco activity if added after fallover had occurred. Gel filtration of the RuBP-rubisco complex to remove unbound RuBP allowed full activation of the enzyme, but the inhibition of activated rubisco during fallover was only partially reversed by gel filtration. Addition of alkaline phosphatase completely restored rubisco activity following fallover. The results suggest that fallover is not caused by binding of RuBP to decarbamylated enzyme, but results from binding of a phosphorylated inhibitor to the active site of rubisco. The inhibitor may be a contaminant in preparations of RuBP or may be formed on the active site but is apparently removed from the enzyme in the presence of the rubisco activase protein.     

Proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase in Citrus leaf extracts

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39) has been purified from orange [ Citrus sinensis (L.) Osbeck cv. Washington Navel] leaves using sucrose gradient centrifugation in a fixed angle rotor. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands corresponding to the two subunits of RuBP carboxylase were found. The large subunit coincided with the polypeptide band that has been previously reported to be preferentially mobilized during the spring and summer flush periods.
The degradation of RuBP carboxylase during autodigestion of Citrus leaf extracts, investigated by SDS-PAGE, occurred mainly at acidic (2.5-5.5) pH. The two subunits showed differences in the rate of degradation, the smaller being more rapidly hydrolyzed than the larger. At least four proteolytic activities were identified by means of inhibitor experiments: 1) a pepstatin A-sensitive activity that acts on both RuBP carboxylase subunits, 2) a mercurial ( p -hydroxymercuribenzoate and p -chloromercuriphenylsulfonate)-sensitive activity that degrades only the small subunit, 3) an EDTA-sensitive activity that hydrolyzes both the large and small subunits, and 4) a mercurial-stimulated activity that acts only on the large subunit. It is suggested that the last two proteases may be responsible for the degradation of RuBP carboxylase observed in vivo during the periods of mobilization of leaf protein in Citrus .     

Questions about the complexity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) has played a central role in our understanding of chloroplast biogenesis and photosynthesis. In particular, its catalysis of the rate-limiting step of CO₂ fixation, and the mutual competition of CO₂ and O₂ at the active site, makes Rubisco a prime focus for genetically engineering an increase in photosynthetic productivity. Although it remains difficult to manipulate the chloroplast-encoded large subunit and nuclear-encoded small subunit of crop plants, much has been learned about the structure/function relationships of Rubisco by expressing prokaryotic genes in Escherichia coli or by exploiting classical genetics and chloroplast transformation of the green alga Chlamydomonas reinhardtii. However, the complexity of chloroplast Rubisco in land plants cannot be completely addressed with the existing model organisms. Two subunits encoded in different genetic compartments have coevolved in the formation of the Rubisco holoenzyme, but the function of the small subunit remains largely unknown. The subunits are posttranslationally modified, assembled via a complex process, and degraded in regulated ways. There is also a second chloroplast protein, Rubisco activase, that is responsible for removing inhibitory molecules from the large-subunit active site. Many of these complex interactions and processes display species specificity. This means that attempts to engineer or discover a better Rubisco may be futile if one cannot transfer the better enzyme to a compatible host. We must frame the questions that address this problem of chloroplast-Rubisco complexity. We must work harder to find the answers.     

Two quick methods for isolation of ribulose-1,5-bisphosphate carboxylase/oxygenase

Methods are described which allow the isolation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (rubisco) in a very short time. Source of the material was highly impure commercial enzyme in the case of spinach rubisco or bacteria grown from a fermentor in the case of Alcaligenes eutrophus rubisco. Purity of the enzymes is demonstrated by gel electrophoreses. Enzyme isolated from fresh cells gave crystals of excellent diffraction, suitable for X-ray structure analyisis.     

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

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis, but O2 competes with CO2 for substrate ribulose 1,5-bisphosphate, leading to the loss of fixed carbon. Interest in genetically engineering improvements in carboxylation catalytic efficiency and CO2/O2 specificity has focused on the chloroplast-encoded large subunit because it contains the active site. However, there is another type of subunit in the holoenzyme of plants, which, like the large subunit, is present in eight copies. The role of these nuclear-encoded small subunits in Rubisco structure and function is poorly understood. Small subunits may have originated during evolution to concentrate large-subunit active sites, but the extensive divergence of structures among prokaryotes, algae, and land plants seems to indicate that small subunits have more-specialized functions. Furthermore, plants and green algae contain families of differentially expressed small subunits, raising the possibility that these subunits may regulate the structure or function of Rubisco. Studies of interspecific hybrid enzymes have indicated that small subunits are required for maximal catalysis and, in several cases, contribute to CO2/O2 specificity. Although small-subunit genetic engineering remains difficult in land plants, directed mutagenesis of cyanobacterial and green-algal genes has identified specific structural regions that influence catalytic efficiency and CO2/O2 specificity. It is thus apparent that small subunits will need to be taken into account as strategies are developed for creating better Rubisco enzymes.     

Proteolytic degradation of barley ribulose-1,5-bisphosphate carboxylase/oxygenase and recognition of the fragments by monoclonal antibodies

Limited proteolysis of barley ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) was effected by treatments with trypsin and Staphylococcus aureus strain V8 protease. Treatment of native RuBPCO with proteases resulted in the degradation of the large subunit (LS) of the enzyme. Trypsin cleaved three fragments from the LS but the S. aureus strain V8 protease cleaved only one. The small subunit (SS) was not affected. In the presence of 0.5 % sodium dodecyl sulfate, RuBPCO degraded into several fragments; some of them were fairly stable. Monoclonal antibodies (Mabs) against barley RuBPCO were applied in immunoblotting analysis to distinguish which of the fragments were recognized. All the Mabs recognized the fragments with molecular masses close to those of the LS. Differences among Mabs were observed in the fragments with low molecular mass. This revised version was published online in June 2006 with corrections to the Cover Date.     

Alteration of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase activase activities by site-directed mutagenesis

Site-directed mutagenesis was performed on the 1.6 and 1.9 kilobase spinach (Spinacea oleracea) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase cDNAs, encoding the 41 and 45 kilodalton (kD) isoforms of the enzyme, to create single amino acid changes in the putative ATP-binding site of Rubisco activase (Lys-107, Gln-109, and Ser-112) and in an unrelated cysteine residue (Cys-256). Replacement of Lys-107 with Met produced soluble protein with reduced Rubisco activase and ATPase activities in both isoforms. Substituting Ala or Arg for Lys-107 produced insoluble proteins. Rubisco activase activity increased in the 41-kD isoform when Gln-109 was changed to Glu, but activity in the 45-kD isoform was similar to the wild-type enzyme. ATPase activity in the Glu-109 mutations did not parallel the changes in Rubisco activase activity. Rather, a higher ratio of Rubisco activase to ATPase activity occurred in both isoforms. The mutation of Gln-109 to Lys inactivated Rubisco activase activity. Replacement of Ser-112 with Pro created an inactive protein, whereas attempts to replace Ser-112 with Thr were not successful. The mutation of Cys-256 to Ser in the 45-kD isoform reduced both Rubisco activase and ATPase activities. The results indicate that the two activities of Rubisco activase are not tightly coupled and that variations in photosynthetic efficiency may occur in vivo by replacing the wild-type enzyme with mutant enzymes.     

Proteolytic degradation of barley ribulose-1,5-bisphosphate carboxylase/oxygenase and recognition of the fragments by monoclonal antibodies

Limited proteolysis of barley ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) was effected by treatments with trypsin and Staphylococcus aureus strain V8 protease. Treatment of native RuBPCO with proteases resulted in the degradation of the large subunit (LS) of the enzyme. Trypsin cleaved three fragments from the LS but the S. aureus strain V8 protease cleaved only one. The small subunit (SS) was not affected. In the presence of 0.5 % sodium dodecyl sulfate, RuBPCO degraded into several fragments; some of them were fairly stable. Monoclonal antibodies (Mabs) against barley RuBPCO were applied in immunoblotting analysis to distinguish which of the fragments were recognized. All the Mabs recognized the fragments with molecular masses close to those of the LS. Differences among Mabs were observed in the fragments with low molecular mass.     

Characteristics of ribulose-1,5-bisphosphate carboxylase/oxygenase degradation by lysates of mechanically isolated chloroplasts from wheat leaves

The lysate from intact chloroplasts mechanically isolated from primary leaves of 9 day old seedlings of wheat (Triticum aestivum L. var Aoba) was incubated in the pH range of 5.5 to 8.5 at 37°C for 5 hours. Proteolytic activity against ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was estimated by disappearance of the large subunit of Rubisco or the appearance of its degradation products. Although the activity in lysates was weak, the products were detected by applying Western blotting. The degradation products were similar to those obtained when Rubisco was incubated with the lysate of vacuoles isolated from like leaves. Although some of the products were similar to those from vacuole lysates, many were clearly different after incubation of Rubisco with trypsin, V-8 protease, or reactive oxygen (hydroxy radical). Lysates of chloroplasts, pretreated with thermolysin at 4°C for 30 minutes, had no proteolytic activity against Rubisco after incubation at 37°C for 5 hours. These results show that the proteolytic activity against Rubisco found in lysates of our mechanically isolated chloroplasts was mostly due to the contamination of vacuolar proteases adhering to the outer envelope of the chloroplasts during their isolation.     

Degradation of active-oxygen-modified ribulose-1,5-bisphosphate carboxylase/oxygenase by chloroplastic proteases requires ATP-hydrolysis

Active oxygen (AO) species generated in plants under stress conditions trigger degradation of Rubisco (EC 4.1.1.39). To find out whether AO species activate proteases or make the protein susceptible to proteolysis, purified and ¹⁴C-labelled Rubisco protein was incubated with stromal preparations obtained from barley (Hordeum vulgare L.) leaves. The protein was degraded into distinct fragments only after a treatment with AO. This result shows that AO-treated Rubisco has been modified to become a substrate for stromal protease(s) and dismisses the possibility of protease activation. Upon degradation, distinct fragments accumulated with time. The fragmentation pattern was indistinguishable from that obtained with intact chloroplasts subjected to oxidative conditions (cf. M. Desimone et al., 1996, Plant Physiol 111: 789–796). Degradation required ATP-hydrolysis, since AMP, ADP or non-hydrolysable ATP-analogs did not support proteolysis. The ClpP-deficient stromal preparations degraded AO-modified Rubisco, making the involvement of the ClpC/P protease unlikely. Received: 1 September 1997 / Accepted 15 November 1997     

The oxygenase activity of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

Catalysis by pure ribulose bisphosphate carboxylase from $\text{Rhodospirillum rubrum}$, which is a dimer (MW: 114,000) lacking small subunits, is inhibited by oxygen. Oxygen is a competitive inhibitor with respect to carbon dioxide. In the absence of carbon dioxide, the enzyme catalyzes the oxygenolytic cleavage of ribulose-1,5-bisphosphate with consumption of one mole of oxygen per mole of 3-phosphoglycerate produced.     

Preliminary structural studies of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from Rhodospirillum rubrum has been crystallized in a form that is suitable for structural studies by x-ray diffraction. The asymmetric unit of the crystal contains one dimeric enzyme molecule of molecular mass 101,000 Da. The enzyme was activated prior to crystallization and is presumed to be in the CO2-activated state in the crystal. The method of hydrophobicity correlation has been used to compare the amino acid sequence of this molecule (466 residues) to that of the large subunit of a higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (477 residues in Nicotiana tabacum). The pattern of residue hydrophobicities is similar along the two polypeptides. This suggests that the three-dimensional folding of the large polypeptide chains may be similar in plant and bacterial enzymes. If this is so, knowing the structure of either the plant or bacterial ribulose-1,5-bisphosphate carboxylase/oxygenase should aid in learning the structure of the other.