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1.
The genes for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) were cloned from a thermophilic cyanobacterium, Synechococcus sp. strain a-1. These two genes were located tandemly on the same strand of chromosomal DNA with a 467 bp spacer region. The rbcL gene codes for 474 amino acid residues (1,422 bp), and rbcS for 118 residues (354 bp). The deduced amino acid sequence for the large subunit was highly similar to those of other cyanobacteria and higher plants. The RubisCO genes were overexpressed (>30% of total soluble protein) in E. coli cells by using pKK223-3 as an expression vector. The overexpressed RubisCO formed hexademers (L8S8 form) in E. coli cells, the same form as in Synechococcus cells. The RubisCO was easily purified from E. coli cell free extract using the advantage of its high thermostability, and the purified RubisCO had almost the same characteristics as the native RubisCO purified from Synechococcus sp. strain a-1.  相似文献   

2.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) rapidly extracted from leaves of wheat (Triticum aestivum) and purified activated RuBPCO were incubated in the presence and absence of 20 millimolar HCO3 and changes in activation state were followed. Rapid inactivation occurred in the presence, but not in the absence, of HCO3. Effects of CO2 concentration and pH during preincubation before assay on activation state of RuBPCO were investigated in equilibrium studies. Twenty percent inactivation occurred at high CO2 concentration if pH was high, but not if it was low, suggesting that RuBPCO was inactivated by HCO3. The inactivation by HCO3 was more rapid than the dissociation of activating CO2 in CO2-free buffer (both in the presence of 20 millimolar MgCl2), suggesting that HCO3 was bound to the active enzyme complex. The dissociation of inactivating HCO3 from the enzyme was slow enough that inhibition could be demonstrated in experiments with HCO3 treatments during preincubation and constant conditions during assay. Inorganic phosphate did not seem to interfere with the binding of HCO3.  相似文献   

3.
Wang ZY  Portis AR 《Plant physiology》1992,99(4):1348-1353
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 (Koff) 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.  相似文献   

4.
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 KD of the enzyme/substrate complex is ≤1 μM, all ribulose-P2 generated in situ may be stored as this complex to prevent decomposition.  相似文献   

5.
6.
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.  相似文献   

7.
When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA).  相似文献   

8.
9.
10.
The arrangement of subunits of ribulosebisphosphate carboxylase in solution has been studied by exposing the enzyme to the cross-linking agents tetranitromethane, dimethyl suberimidate, and dimethyl adipimidate, and the cleavable cross-linking agent, methyl 4-mercaptobutyrimidate followed by gel electrophoresis in the presence of dodecyl sulfate. All these agents caused the formation of dimers of the enzyme's small subunit, independently of protein concentration. In addition, trimers and tetramers of small subunit were detected in the mercaptobutyrimidate-treated enzyme. The data show that small subunits are closely paired in the native enzyme and may be in layers of four, or a ring of eight.  相似文献   

11.
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13.
Intermediates in the ribulose-1,5-bisphosphate carboxylase reaction   总被引:2,自引:0,他引:2  
At least two intermediates of the D-ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) reaction were liberated in detectable amounts when the functioning enzyme from Rhodospirillum rubrum was quenched in acid. Using substrate labeled with 32P in C-1, [32P]orthophosphate (Pi) was found when the quenched solution was rapidly processed for extraction of Pi as the acid molybdate complex. Reaction with sodium borohydride under mildly alkaline conditions immediately after acid quenching of the carboxylase reaction decreased the amount of 32Pi that was observed by 68%. The compound whose degradation to Pi was prevented by reaction with sodium borohydride decomposed under both acid and neutral conditions with a half-time of about 5 min at 25 degrees C and was assigned to the beta-keto acid recently demonstrated for the spinach enzyme ( Schloss , J.V., and Lorimer , G.H. (1982) J. Biol. Chem. 257, 4691-4694). It was sufficiently stable upon neutralization to react productively with fresh enzyme. As substrate CO2 concentration was decreased below the steady state Km value, the proportion of the 32P that did not react with sodium borohydride increased, indicative of a second unstable intermediate that precedes the carboxylation step. The decomposition of the latter intermediate to Pi, which occurs with a t1/2 less than or equal to 6 ms, was prevented if I2 was present in the acid quench medium. These are properties expected of the 2,3- enediol form of ribulose bisphosphate. Both intermediates reach their maximum levels when product formation is most rapid and disappear when product formation is complete as expected of reaction intermediates.  相似文献   

14.
Catalysis by pure ribulose bisphosphate carboxylase from 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.  相似文献   

15.
Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.  相似文献   

16.
The three-dimensional structure of the complex of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum, CO2, Mg2+, and ribulose bisphosphate has been determined with x-ray crystallographic methods to 2.6-A resolution. Ribulose-1,5-bisphosphate binds across the active site with the two phosphate groups in the two phosphate binding sites of the beta/alpha barrel. The oxygen atoms of the carbamate and the side chain of Asp-193 provide the protein ligands to the bound Mg2+ ion. The C2 and the C3 or C4 oxygen atoms of the substrate are also within the first coordination sphere of the metal ion. At the present resolution of the electron density maps, two slightly different conformations of the substrate, with the C3 hydroxyl group "cis" or "trans" to the C2 oxygen, can be built into the observed electron density. The two different conformations suggest two different mechanisms of proton abstraction in the first step of catalysis, the enolization of the ribulose 1,5-bisphosphate. Two loop regions, which are disordered in the crystals of the nonactivated enzyme, could be built into their respective electron density. A comparison with the structure of the quaternary complex of the spinach enzyme shows that despite the different conformations of loop 6, the positions of the Mg2+ ion, and most atoms of the substrate are very similar when superimposed on each other. There are, however, some significant differences at the active site, especially in the metal coordination sphere.  相似文献   

17.
Crystals from the dimeric enzyme ribulose-1,5-bisphosphate carboxylase of the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. The crystals are of the quarternary complex comprising enzyme: activator CO2 (as a carbamate): Mg2+: 2- carboxyarabinitol -1,5-bisphosphate (as a transition state analog). X-ray diffraction photographs show symmetry consistent with space group P4(1)2(1)2 or the corresponding enantiomorphic space group. Cell parameters are a = b = 82 A, c = 324 A with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution.  相似文献   

18.
《Plant science》1986,44(2):119-123
The low activity of ribulose bisphosphate carboxylase from darkened soybean (Glycine max [L.] Merr. cv. Bragg) leaves was not raised to the level of that from leaves in the light by CO2 and Mg2+, even after a 4-h incubation. The extract of darkened leaves, unlike the extract from illuminated leaves, was not fully CO2/Mg2+-activatable after Sephadex gel filtration in the absence of Mg2+. (NH4)2SO4 fractionation eliminated the inhibition effect found in the dark extracts resulting in similar rates for the extracts obtained from leaves in the dark and light. Although the Vmax values of the gel-filtered extracts from dark and light leaves differed by 3-fold, the Km(CO2)-values were the same (12.7 μM), as were the Km(RuBP)-values (250 μM). These data support the hypothesis that for soybean leaves in the dark a tightly-binding inhibitor renders much of the ribulose bisphosphate carboxylase enzyme catalytically non-functional.  相似文献   

19.
Wheat ribulose-1,5-diphosphate carboxylase purified to homogeneity had a MW of 540 000, sedimentation coefficient (S20, W) of 18.5 S, apparent diffusion constant (Dapp) of 3.07 × 10?7 cm2/sec, Stoke's radius 5.44 nm, and fractional ratio of 1.17. Electron microscopy revealed particles of 10–12 nm diameter. The enzyme was dissociated by sodium dodecyl sulphate into two subunits of MW 53 000 (S20, W = 3.0 S) and 13 500 (S20, W = 1.7 S). The total amino acid residues in the large and small subunits were 481 and 117, respectively. Tryptic peptide maps of the two subunits confirmed the estimated numbers of Arg and Lys residues. Although the amino acid pattern of the large subunit closely resembled that from barley, rather than that for spinach, beet or tobacco, the pattern of the small subunit was markedly different from those of all the other species.  相似文献   

20.
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.  相似文献   

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