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.     

Enzymic Properties of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Purified from Rice Leaves

The enzymic properties of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase purified from rice (Oryza sativa L.) leaves were studied. Rice RuBPcarboxylase, activated by preincubation with CO₂ and Mg²⁺ like other higher plant carboxylases, had an activation equilibrium constant (K_cK_Mg) of 1.90 × 10⁵ to 2.41 × 10⁵ micromolar² (pH 8.2 and 25°C). Kinetic parameters of carboxylation and oxygenation catalyzed by the completely activated enzyme were examined at 25°C and the respective optimal pHs. The K_m(CO₂), K_m(RuBP), and V_max values for carboxylation were 8 micromolar, 31 micromolar, and 1.79 units milligram⁻¹, respectively. The K_m(O₂), K_m(RuBP), and V_max values for oxygenation were 370 micromolar, 29 micromolar, and 0.60 units milligram⁻¹, respectively.

Comparison of rice leaf RuBP carboxylase with other C₃ plant carboxylases showed that it had a relatively high affinity for CO₂ but the lowest catalytic turnover number (V_max) among the species examined.

     

Interaction of Hydrogen Peroxide with Ribulose-1,5-bisphosphate Carboxylase/Oxygenase from Rice

The properties of rice-derived ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in different concentrations of hydrogen peroxide (H2O2) solutions have been studied. The results indicate that at low H2O2 concentrations (0.2-10 mM), the properties of rubisco (e.g., carboxylase activities, structure, and susceptibility to heat denaturation) change slightly. However, at higher H2O2 concentrations (10-200 mM), rubisco undergoes an unfolding process, including the loss of secondary and tertiary structure, forming extended hydrophobic interface, and leading to cross-links between large subunits. High concentrations of H2O2 can also result in an increase in susceptibility of rubisco to heat denaturation. Further pre-treatments with or without reductive reagents to rubisco show that the disulfide bonds in rubisco help to protect the enzyme from damage by H2O2 as well as other reactive oxygen species.     

烟草核酮糖-1,5-二磷酸羧化酶/加氧酶四级结构的研究(英文)

本文提出三种证据证明烟草核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)的大亚基伸展在小亚基的外面,小亚基排列在大亚基中间的概念。证据是:1.固定化胰蛋白酶在一定条件下可水解RubisCO的大亚基但不水解小亚基,而天然胰蛋白酶水解大亚基,也水解小亚基。2.固定化抗小亚基IgG-Sepharose可与游离的小亚基相结合,但不能与全酶结合。3.低浓度尿素处理可使固定化的RubisCO-Sepharose上的小亚基解离下来,而大亚基仍结合在载体上,这说明RubisCO是通过定位在分子表面上的大亚基的ε-氨基与Sepharose共价偶联的。当RubisCO中的小亚基全部被解离后,大亚基之间的结合进一步增强,这时解离大亚基所需的尿素浓度要比小亚基存在时高。任何RubisCO的四级结构模型都应将小亚基置于大亚基中间受保护的位置,一部份小亚基可暴露于全酶分子表面。     

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

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

The Intracellular Localization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Chlamydomonas reinhardtii

The pyrenoid is a proteinaceous structure found in the chloroplast of most unicellular algae. Various studies indicate that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in the pyrenoid, although the fraction of Rubisco localized there remains controversial. Estimates of the amount of Rubisco in the pyrenoid of Chlamydomonas reinhardtii range from 5% to nearly 100%. Using immunolocalization, the amount of Rubisco localized to the pyrenoid or to the chloroplast stroma was estimated for C. reinhardtii cells grown under different conditions. It was observed that the amount of Rubisco in the pyrenoid varied with growth condition; about 40% was in the pyrenoid when the cells were grown under elevated CO₂ and about 90% with ambient CO₂. In addition, it is likely that pyrenoidal Rubisco is active in CO₂ fixation because in vitro activity measurements showed that most of the Rubisco must be active to account for CO₂-fixation rates observed in whole cells. These results are consistent with the idea that the pyrenoid is the site of CO₂ fixation in C. reinhardtii and other unicellular algae containing CO₂-concentrating mechanisms.     

Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO

The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO₂ fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg²⁺ for catalysis and, like RuBisCO, was stimulated by CO₂. Four residues that are essential for the enolization reaction of RuBisCO, Lys¹⁷⁵, Lys²⁰¹, Asp²⁰³, and Glu²⁰⁴, were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys¹²³, the residue conserved in DK-MTP-1-P enolases, was also essential for B. subtilis RLP enolase activity. Similarities between the active site structures of RuBisCO and B. subtilis RLP were examined by analyzing the effects of structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in the DK-MTP-1-P enolase reaction with a K_i value of 103 μm. RuBP and d-phosphoglyceric acid, the substrate and product, respectively, of RuBisCO, were weaker competitive inhibitors. These results suggest that the amino acid residues utilized in the B. subtilis RLP enolase reaction are the same as those utilized in the RuBisCO RuBP enolization reaction.Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)⁴ catalyzes the carboxylation and oxygenation reactions of ribulose 1,5-bisphosphate (RuBP) in photosynthesis (1-4). This enzyme is the sole CO₂-fixing enzyme in plants; however, it has certain inefficiencies. It has a very low turnover rate, a low affinity for the substrate, CO₂, and low specificity between the carboxylation and oxygenation reactions (5-7). Thus, the intrinsic enzymatic properties of RuBisCO are inadequate for efficient incorporation of CO₂ into organic matter in photosynthesis (7). However, plants have overcome these disadvantages by investing a huge amount of leaf nitrogen in RuBisCO synthesis (8).In nature, there are wide variations in the properties and primary sequences of RuBisCO among different photosynthetic organisms (9-12). The primary sequences vary as much as 73% without loss of activity. The relative specificity ranges from ∼0.5 in a small subunitless RuBisCO to 238 in a red algal, hexadecameric RuBisCO (13, 14). The affinity for CO₂ varies some 100-fold (15). Comparisons between these kinetic parameters and the primary sequences are expected to reveal promising strategies for improving the enzyme, and many studies have been conducted on this topic (7, 16-18).A RuBisCO-like protein (RLP) with no CO₂-fixing activity was first demonstrated in Chlorobium tepidum (19), and a similar protein in Bacillus subtilis was found to be involved in the methionine salvage pathway (20). These findings have pointed to a new direction in RuBisCO research (17, 21). The phylogenetic tree of the catalytic subunits of RuBisCOs and their homologs shows four major clusters, forms I-III, and form IV (Fig. 1A). Form I and II RuBisCOs are involved in photosynthetic or chemosynthetic CO₂ fixation, whereas the metabolic function of form III RuBisCOs remains unclear, although they can fix CO₂ on RuBP (9, 22). Forms I-III conserve almost all 19 amino acid residues that are essential for CO₂ fixation in RuBisCO (Fig. 1B). The form IV cluster in the phylogenetic tree consists of RLPs that show ∼20% homology to plant form I or bacterial form II RuBisCOs (12, 20, 21, 23-25). There are 8-18 RuBisCO-essential residues that are conserved in RLPs (Fig. 1B). Form IV RLPs are further subdivided into four groups; α1, α2, β, and γ (21). The RLP of B. subtilis is classified in α1 and catalyzes the enolization reaction of 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) but not the carboxylation of RuBP (Fig. 2A) (20, 21, 23). The absence of CO₂-fixing activity in the B. subtilis RLP may be ascribed to changes in 8 of the 19 amino acid residues essential for CO₂ fixation in RuBisCO (Fig. 1B). Several of these residues are located at the C-terminal domains of B. subtilis RLP and RuBisCO. The dimeric RuBisCO from Rhodospirillum rubrum catalyzes the DK-MTP-1-P enolase reaction with very low activity (20). These findings, together with the similarity in the chemical structures of substrates for B. subtilis RLP and RuBisCO (Fig. 2A), suggest that they may have a close evolutionary relationship (12, 21, 23-25).Open in a separate windowFIGURE 1.Homology between RLPs and RuBisCOs. A, phylogenetic tree of RLPs and RuBisCOs. Deduced amino acid sequence of B. subtilis subsp. subtilis str. 168 RLP ({"type":"entrez-protein","attrs":{"text":"NP_389242","term_id":"16078423","term_text":"NP_389242"}}NP_389242) was compared with sequences of RLPs of Thermotoga lettingae TMO ({"type":"entrez-protein","attrs":{"text":"YP_001471302","term_id":"157364535","term_text":"YP_001471302"}}YP_001471302), Beggiatoa sp. SS ({"type":"entrez-protein","attrs":{"text":"ZP_01997270","term_id":"153864333","term_text":"ZP_01997270"}}ZP_01997270), Ostreococcus tauri (Ostreococcus tauri IV, {"type":"entrez-protein","attrs":{"text":"CAL54998","term_id":"116059291","term_text":"CAL54998"}}CAL54998), Alkalilimnicola ehrlichei MLHE-1 ({"type":"entrez-protein","attrs":{"text":"YP_742007","term_id":"114320324","term_text":"YP_742007"}}YP_742007), R. rubrum ATCC 11170 (R. rubrum IV, {"type":"entrez-protein","attrs":{"text":"YP_427085","term_id":"83593333","term_text":"YP_427085"}}YP_427085), R. palustris CGA009 (R. palustris IV-1, {"type":"entrez-protein","attrs":{"text":"NP_947514","term_id":"39935238","term_text":"NP_947514"}}NP_947514), Archaeoglobus fulgidus DSM 4304 (A. fulgidus IV, {"type":"entrez-protein","attrs":{"text":"NP_070416","term_id":"11499182","term_text":"NP_070416"}}NP_070416), M. aeruginosa PCC 7806 (M. aeruginosa IV, {"type":"entrez-protein","attrs":{"text":"CAJ43366","term_id":"112821115","term_text":"CAJ43366"}}CAJ43366), G. kaustophilus HTA426 ({"type":"entrez-protein","attrs":{"text":"YP_146806","term_id":"56419488","term_text":"YP_146806"}}YP_146806), Bacillus cereus ATCC 14579 ({"type":"entrez-protein","attrs":{"text":"NP_833754","term_id":"30022123","term_text":"NP_833754"}}NP_833754), B. bronchiseptica RB50 ({"type":"entrez-protein","attrs":{"text":"NP_887583","term_id":"33600023","term_text":"NP_887583"}}NP_887583), Polaromonas sp. JS666 ({"type":"entrez-protein","attrs":{"text":"YP_546958","term_id":"91786006","term_text":"YP_546958"}}YP_546958), C. tepidum TLS ({"type":"entrez-protein","attrs":{"text":"NP_662651","term_id":"21674586","term_text":"NP_662651"}}NP_662651), and R. palustris CGA009 (R. palustris IV-2, {"type":"entrez-protein","attrs":{"text":"NP_945615","term_id":"39933339","term_text":"NP_945615"}}NP_945615) and of RuBisCOs of R. palustris CGA009 (R. palustris II, {"type":"entrez-protein","attrs":{"text":"NP_949975","term_id":"39937699","term_text":"NP_949975"}}NP_949975), R. rubrum ATCC 11170 (R. rubrum II, {"type":"entrez-protein","attrs":{"text":"YP_427487","term_id":"83593735","term_text":"YP_427487"}}YP_427487), M. jannaschii DSM 2661 ({"type":"entrez-protein","attrs":{"text":"NP_248230","term_id":"15669420","term_text":"NP_248230"}}NP_248230), A. fulgidus DSM 4304 (A. fulgidus III, {"type":"entrez-protein","attrs":{"text":"NP_070466","term_id":"11499228","term_text":"NP_070466"}}NP_070466), Thermococcus kodakaraensis KOD1 ({"type":"entrez-protein","attrs":{"text":"YP_184703","term_id":"57642225","term_text":"YP_184703"}}YP_184703), Galdieria partita ({"type":"entrez-protein","attrs":{"text":"BAA75796","term_id":"4519903","term_text":"BAA75796"}}BAA75796), R. palustris CGA009 (R. palustris I, {"type":"entrez-protein","attrs":{"text":"NP_946905","term_id":"39934629","term_text":"NP_946905"}}NP_946905), M. aeruginosa PCC 7806 (M. aeruginosa I, {"type":"entrez-protein","attrs":{"text":"CAJ43363","term_id":"112821111","term_text":"CAJ43363"}}CAJ43363), O. tauri (O. tauri IV, {"type":"entrez-protein","attrs":{"text":"YP_717262","term_id":"113170470","term_text":"YP_717262"}}YP_717262), and S. oleracea ({"type":"entrez-protein","attrs":{"text":"NP_054944","term_id":"11497536","term_text":"NP_054944"}}NP_054944). When an organism has more than one RuBisCO and/or RLP sequence, the form number of each sequence in the RuBisCO family follows the name of the organism. ClustalW and TreeView programs (available on the World Wide Web) were used to construct the phylogenetic tree. B, multiple alignments of sequences underlined in A. Identical amino acid residues are indicated by black shading, and similar amino acid residues are indicated by gray shading. Sequences are numbered according to the S. oleracea sequence. Catalytic and RuBP-binding residues deduced for RuBisCO are indicated by open triangles and filled triangles, respectively. Alignment was visualized with the BOXSHADE program (available on the World Wide Web).Open in a separate windowFIGURE 2.Catalytic and structural similarity of RLPs and RuBisCOs. A, catalytic reactions of RuBisCO and RLP. B, comparison of active sites between S. oleracea RuBisCO binding CABP (8RUC) and G. kaustophilus RLP (2OEM) modeled to bind DK-MTP-1-P. DK-MTP-1-P in G. kaustophilus RLP was depicted by substituting the methyl group of DK-H-1-P in 2OEM with the thiomethyl group of MTRu-1-P bound to MtnA (28). Side chains of active site residues and ligands are shown as sticks. These five residues of B. subtilis were substituted with other amino acids in this study. CABP and DK-MTP-1-P are shown in white, and their phosphate groups are shown in red and orange, respectively. Mg²⁺ atoms are shown in yellow. Protein structures were drawn with PyMOL (available on the World Wide Web).The RuBisCO reaction starts with the abstraction of the C3 proton from RuBP to form the cis-enediol(ate) of RuBP (Fig. 2A) (26). Using the spinach numbering format to identify RuBisCO and RLP residues, the carbamate formed on the ε-amino group of Lys²⁰¹ may be the general base to abstract the proton, and the cis-enediol(ate) form of RuBP is stabilized in the combination of side chains from Lys¹⁷⁵ and His²⁹⁴ (27). Asp²⁰³, Glu²⁰⁴, and the carbamate Lys²⁰¹ of the enzyme active site stabilize the cis-enediol(ate) and CO₂ through the Mg²⁺ ion (26). The B. subtilis RLP abstracts the C1 proton of its substrate DK-MTP-1-P to start the DK-MTP-1-P enolization reaction (12, 21, 23). The ε-amino group of Lys¹²³ is thought to be required for the abstraction of the 1-proS proton in the Geobacillus kaustophilus RLP, which belongs to group α1, together with the B. subtilis RLP (Fig. 2B) (25). Lys¹²³ is conserved among DK-MTP-1-P enolases and resides very near the C1 of 2,3-diketohexane 1-phosphate (DK-H-1-P), a structural analogue of DK-MTP-1-P. As is the case in RuBisCO, the enolate intermediate is stabilized by Mg²⁺ and several amino acid residues: Lys¹⁷⁵, Asp²⁰³, Glu²⁰⁴, His²⁹⁴, and the carbamylated Lys²⁰¹.The results of these studies suggest that the DK-MTP-1-P enolase is structurally and functionally related to photosynthetic RuBisCO. However, research on the G. kaustophilus RLP revealed that the proton-abstracting, reaction-starting residues differed between the DK-MTP-1-P enolase and RuBisCO (25). It has been reported that when lysine at 201 is substituted with an alanine in the G. kaustophilus RLP, the enzyme is still capable of catalyzing enolization of DK-MTP-1-P (25). This result raises a question about the above hypothesis on the close evolutionary relationship between the RLP and RuBisCO, because a carbamylated lysine residue would be required at this position to form the Mg²⁺-chelating triad linkage together with Asp²⁰³ and Glu²⁰⁴ and to stabilize the reaction intermediate in the RuBP enolization reaction of RuBisCO.Evolutionary relationships of genes with similar sequences are deduced by comparing gene sequence homology of the genes and amino acid sequence homology of the predicted proteins and by analyzing conservation of functional motifs of the predicted proteins in silico. Comparison of protein structures at the active sites also provides important information. However, it may difficult to predict their mutual evolutionary relationship more precisely when they catalyze different reactions in individual metabolic pathways. The present research adopted a new method to resolve such an issue.We studied the structural and functional interrelationships of RLP and RuBisCO after enzymological characterization of B. subtilis RLP as the DK-MTP-1-P enolase enzyme. The results showed that DK-MTP-1-P enolase activity was limited to some RLPs in the cluster, including B. subtilis in form IV RLPs. All of the catalytic residues for the RuBisCO reaction were also indispensable for DK-MTP-1-P enolase activity. The architecture of the B. subtilis RLP substrate-binding residues stereospecifically stabilized the transition state analog in CO₂ fixation of RuBisCO. The fact that the transition state analog of RuBisCO interacts with the active site of Bacillus RLP strongly supports their evolutionary proximity.     

Measurement of the Concentration of the Active Centers of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in vivo

Methods for in vivo measurement of the concentration of the reactive centers of ribulose-1,5,-bisphosphate carboxylase/oxygenase (Rubisco) are suggested that are based on saturation of the active centers with RuBP and determination of the concentration of the Rubisco–RuBP complex. The total concentration of potentially reactive centers is calculated from the dependence of the concentration of this complex on CO₂ concentration at a steady-state photosynthetic rate with further extrapolation of the carbon dioxide dependence curve to a zero CO₂ concentration. The concentration of centers that possessed a catalytic activity under given environmental conditions was measured after transferring leaves having a steady-state photosynthetic rate into a medium devoid of CO₂ and O₂. This procedure ensured the saturation of the carboxylation centers with RuBP. The carboxylation rates were measured during a short-term exposure to ¹⁴CO₂, and the concentration of the complex was calculated using the values of CO₂ concentration during the exposure time, as well as the carboxylation rate and constant. Rubisco activity was found to decrease at elevated CO₂ concentrations due to a lower concentration of catalytically active enzyme centers.     

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in vivo: Control by High CO2 Concentration

High CO₂ concentrations (HC) in air induce partial deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC 4.1.1.39). Under saturating irradiance, increase in [CO₂] to 1 200 cm³ m^–3 reduces the concentration of operating carboxylation centres by 20–30 %. At a further increase in [CO₂], the activity remained on the same level. Under limiting irradiance, the lowest activity was reached at 600 cm³(CO₂) m^–3. The presence of oxygen diminished deactivation, but O₂ failed to stimulate reactivation under high CO₂. Conditions that favour oxygenation of ribulose-1,5-bisphosphate (RuBP) facilitated reactivation. Even HC did not act as an inhibitor. HC induces deactivation of RuBPCO by increasing the concentration of free reaction centres devoid of the substrate, which are more vulnerable to inhibition than the centres filled with substrates or products.     

A Sensitive Assay Procedure for Simultaneous Determination of Ribulose-1,5-bisphosphate Carboxylase and Oxygenase Activities

A sensitive assay procedure is described for the simultaneous determination of ribulose-1,5-bisphosphate (RuBP) carboxylase and oxygenase activities. In this assay, [1-³H]RuBP is incubated with ¹⁴CO₂ and O₂. Carboxylation rate is determined from ¹⁴CO₂ incorporation and oxygenation rate is determined from [2-³H]glycolate-phosphate production. The assay was found to be suitable at all CO₂ and O₂ concentrations examined, which ranged from 0 to 300 micromolar CO₂ (20 millimolar NaHCO₃) and 0 to 1.15 millimolar (100%) O₂. In combination with a polarographic assay, the stoichiometry of the RuBP oxygenase reaction was found to be RuBP-O₂-glycolate phosphate-glycerate phosphate (1:1:1:1).     

Induction of the Inactivation and Degradation of Phosphoenolpyruvate Carboxylase and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in Maize Leaves by Freezing and Thawing

When frozen leaves of 24-day-old maize (Zea mays L.) plant werethawed on moist filter paper at 26°C (freeze-thaw treatment)several enzymes, including phosphoenolpyruvate carboxylase (PEPC)and ribulose-1,5-bisphosphate carboxylase (RuBPC), were rapidlyinactivated and degraded. The kinetics of the inactivation anddegradation were pseudo first-order, and the halftimes for inactivationof PEPC and RuBPC were 3.2 and 2.4 min, respectively. The effectof the freeze-thaw treatment on the inactivation and degradationdiffered among various enzymes: the residual activities of RuBPC,PEPC, hydroxypyruvate reductase, Cyt c oxidase, NADP-malic enzymeand a-mannosidase 10 min after the start of the thawing treatmentwere 7, 16, 54, 64, 97 and 98% of the initial respective levels.Thirty min after the starting of thawing treatment, the amountsof total soluble protein, the large subunit of RuBPC, the smallsubunit of RuBPC, the PEPC subunit and the NADP-malic enzymesubunit had fallen to 61, 2, 16, 8, and 66% of the initial respectiveamounts. The effect of freeze-thaw treatment on PEPC was greater in oldleaves than in young leaves. There was a steady increase ofthe rate of degradation of PEPC by freeze-thaw treatment asplants aged from 6 to 24 days. These results are discussed inthe context of protein degradation in plant cells. (Received August 9, 1993; Accepted January 10, 1994)     

核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)

文章就核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)的分布、结构、性质、分类与功能的研究进展作了介绍。     

Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30°C, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40°C. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45°C. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40°C. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40°C for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.     

Differential Modulation of Carboxylase and Oxygenase Activities of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Released from Freshly Ruptured Spinach Chloroplasts

The carboxylase activity of ribulose 1,5-bisphosphate (RuBP)carboxylase/oxygenase released from freshly ruptured spinachchloroplasts was stimulated preferentially by Mg²⁺ while oxygenaseactivity was higher with Mn²⁺. Only Mg²⁺ could reactivate eitheractivity of desalted enzyme. The results suggest that carboxylaseand oxygenase activities of RuBP craboxylase/oxygenase can bemodulated selectively by Mg²⁺ or Mn²⁺. ¹ Present address: Department of Botany, Sri Venkateswara University,Tirupati 517 502, India. (Received March 5, 1981; Accepted June 26, 1981)     

RubisCO的研究进展

1,5-二磷酸核酮糖羧化酶/加氧酶(RubisCO)是调节光合和光呼吸,决定净光合作用的一个关键酶;也是植物可溶性蛋白质中含量最高的蛋白质.该酶广泛存在于植物及一些微生物体内.综述了近年来有关RubisCO的一些研究进展. 包括RubisCO的基本性质、结构与功能、酶基因工程、酶活性调节及其活化酶等.     

Ribulose-1,5-bisphosphate Carboxylase/Oxygenase and Polyphenol Oxidase in the Tobacco Mutant Su/su and Three Green Revertant Plants

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) was crystallized from a heterozygous tobacco (Nicotiana tabacum L.) aurea mutant (Su/su), its wild-type sibling (su/su), and green revertant plants regenerated from green spots found on leaves of haploid Su plants. No differences were found in the specific activity or kinetic parameters of this enzyme, when comparing Su/su and su/su plants of the same age, which had been grown under identical conditions. The enzyme crystallized from revertant plants was also identical to the enzyme from wild-type plants with the exception of one clone, designated R2. R2 has a chromosome number approximately double that of the wild-type (87.0 ± 11.1 versus 48). The enzyme from R2 had a lower V_max for CO₂, although the K_m values were identical to those for the enzyme from the wild-type plant. The enzyme from all mutant plants had identical isoelectric points, identical molecular weight as demonstrated by migration on native and sodium dodecyl sulfate (SDS)-polyacrylamide gels, and the same ratio of large to small subunits as the enzyme from the wild-type. The large subunit of the enzyme from tobacco leaves exhibited a different electrophoretic pattern than did the large subunit from spinach; there were two to three bands on SDS-polyacrylamide gels for the tobacco enzyme whereas the enzyme from spinach had only one species of large subunit.