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1.
Homogeneous D-ribulose 1,5-bisphosphate carboxylase from Rhodospirillum rubrum, Chlamydomonas reinhardtii, and Hydrogenomonas eutropha are inhibited by low concentrations of pyridoxal 5′-phosphate. In the case of the enzyme from Rhodospirillum rubrum, this inhibition is strongly antagonized by the substrate, D-ribulose 1,5-bisphosphate. These results suggest that pyridoxal 5′-phosphate may act close to or at the ribulose 1,5-bisphosphate binding site of the enzyme from Rhodospirillum rubrum.  相似文献   

2.
Ribulose-l,5-bisphosphate carboxylase (E.C. 4.1.1.39) isolated from Chromatium strain D contains 64 free cysteinyl -SH groups per mol (Mr 5.11 × 105) as determined using three different titrants: p-[14C]chloromercuribenzoate, the Ellman reagent, and [14C]iodoacetamide.Distribution of -SH groups in the two constituent subunits (A and B) isolated from spinach and Chromatium ribulose-1,5-bisphosphate carboxylases was determined to be for spinach, 9 in A and 3 in B; and for Chromatium, 7 in A and 1 in B.The relationship between the numbers of -SH groups blocked vs residual activities of both the ribulose-1,5-bisphosphate carboxylase and oxygenase reactions was examined by titration with p-chloromercuribenzoate. In both spinach and Chromatium enzymes, antisigmoidal curves were obtained for the degree of the enzyme activity loss in relation to the numbers of -SH groups masked. However, at alkaline pH the Chromatium enzyme shows a sharp decline in both carboxylase and oxygenase activities, apparently due to the alkali dissociation of the enzyme molecule accompanied by its structural deformation. The functional role of -SH groups in the ribulose-1,5-bisphosphate carboxylase molecule is discussed in relation to two constituent enzyme reactions, and it is concluded that in both enzyme sources the active sites are probably the same for the two reactions.  相似文献   

3.
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   

4.
2-Carboxy-d-hexitol 1,6-bisphosphate (CHBP) has been prepared from d-fructose 1,6-bisphosphate and cyanide. DEAE-Sephadex chromatography separated the reaction products into two fractions which were identified as CHBP and CHBP-lactone. CHBP is presumably a mixture of two diastereomers, 2-carboxy-d-glucitol 1,6-bisphosphate and 2-carboxy-d-mannitol 1,6-bisphosphate, but an attempt to separate these compounds was not successful. The material in the CHBP-lactone peak had no effect on d-ribulose 1,5-bisphosphate (RuBP) carboxylase. However, CHBP was a potent reversible inhibitor of RuBP carboxylases. This compound displayed an inhibition constant (Ki at pH 8.0 and 30 °C) of 1–2 μm with the enzymes from spinach and barley, while the Ki was 60–70 μm with bacterial RuBP carboxylases from Pseudomonas oxalaticus and Rhodospirillum rubrum. The mode of inhibition was competitive with respect to RuBP for all the carboxylases, and noncompetitive with respect to CO2 for the enzymes from spinach, P. oxalaticus and R. rubrum. The results indicate that, in the binding of certain organic phosphates by RuBP carboxylases, there may be a fundamental difference between the enzymes isolated from microbial and from higher plant sources. RuBP oxygenase activities from spinach and P. oxalaticus were also inhibited by CHBP, with Ki values which were similar to those obtained with the carboxylase activity of the same enzymes. The mode of inhibition of the oxygenase activities was also competitive with respect to RuBP. Thus, it seems that the binding of CHBP is similar for the carboxylase and oxygenase reactions of the same enzyme.  相似文献   

5.
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 Km values of 10 micromolar for S-adenosyl-l-methionine and 18 micromolar for ribulose-P2 carboxylase, a Vmax 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.  相似文献   

6.
The catalytically active oligomeric form of the larger subunit, Am, obtained from spinach leaf ribulose-1,5-diphosphate carboxylase by pretreatment with p-mercuribenzoate at pH 7.5 followed by incubation at pH 9.0, was free of the smaller subunit based on C-terminal amino acid analyses. Valine was the predominant C-terminus of the Am preparations, the release of tyrosine being negligibly small [cf. Sugiyama and Akazawa, Biochemistry 9 (1970) 4499]. The pH optimum of the ribulose-1,5-diphosphate carboxylase reaction by Am was about 8.5, in comparison to the native enzyme which showed an alkaline pH optimum only in the absence of Mg2+. The substrate saturation curve of the catalytic subunit with respect to bicarbonate followed the Michaelis-Menten equation, as contrasted to the anomalous reaction kinetics of the native ribulose-1,5-diphosphate carboxylase molecule reported previously. These overall results indicate that the allosteric properties of spinach ribulose-1,5-diphosphate carboxylase are possibly conveyed by a unique structural conformation that requires the presence of the smaller subunit in association with the larger catalytic subunit component of the enzyme molecule.  相似文献   

7.
Under mild conditions, 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate rapidly and irreversibly inactivates ribulosebisphosphate carboxylase from Rhodospirillum rubrum. The substrate ribulosebisphosphate protects the enzyme against inactivation. Incorporation of reagent has been quantitated by reduction of the modified carboxylase with [3H]NaBH4. Based on the difference in the levels of incorporation found in the inactivated enzyme as compared with the protected enzyme, loss of enzymic activity results from the modification of about 0.4 residue per catalytic subunit. Analyses of hydrolysates demonstrate that both cysteinyl and lysyl derivatives are present in the inactivated carboxylase; the protected sample contains about the same amount of modified cysteine but little of the modified lysine. Thus, inactivation appears to correlate with derivatization of lysyl residues.  相似文献   

8.
Carbon isotope fractionation by structurally and catalytically distinct ribulose-1,5-bisphosphate carboxylases from one eucaryotic and four procaryotic organisms has been measured under nitrogen. The average fractionation for 40 experiments was −34.1 ‰ with respect to the δ13C of the dissolved CO2 used, although average fractionations for each enzyme varied slightly: spinach carboxylase, −36.5 ‰; Hydrogenomonas eutropha, −38.7 ‰; Agmenellum quadruplicatum, −32.2 ‰; Rhodospirillum rubrum, −32.1 ‰; Rhodopseudomonas sphaeroides peak I carboxylase, −31.4 ‰; and R. sphaeroides peak II carboxylase, −28.3 ‰. The carbon isotope fractionation value was largely independent of method of enzyme preparation, purity, or reaction temperature, but in the case of spinach ribulose-1,5-bisphosphate carboxylase fractionation, changing the metal cofactor used for enzyme activation had a distinct effect on the fractionation value. The fractionation value of −36.5 ‰ with Mg2+ as activator shifted to −29.9 ‰ with Ni2+ as activator and to −41.7 ‰ with Mn2+ as activator. These dramatic metal effects on carbon isotope fractionation may be useful in examining the catalytic site of the enzyme.  相似文献   

9.
The functions of His291, His295 and His324 at the active-site of recombinant A. nidulans ribulose-1,5-bisphosphate carboxylase/ oxygenase have been explored by site-directed mutagenesis. Replacement of His291 by K or R resulted in unassembled proteins, while its replacement by E, Q or N resulted in assembled but inactive proteins. These results are in accord with a metal ion-binding role of this residue in the activated ternary complex by analogy to x-ray crystallographic analyses of tobacco and spinach enzymes.His324 (H327 in spinach), which is located within bonding distance of the 5-phosphate of bound bi-substrate analog 2-carboxyarabinitol 1,5-bisphosphate in the crystal structures, has been substituted by A, K, R, Q and N. Again with the exception of the H324K and R variants, these changes resulted in detectable assembled protein. The mutant H324A protein exhibited no detectable carboxylase activity, whereas the H324Q and H324N changes resulted in purifiable holoenzyme with 2.0 and 0.1% of the recombinant wild-type specific carboxylase activity, respectively. These results are consistent with a phosphate binding role for this residue.The replacement of His295, which has been suggested to aid in phosphate binding, with Ala in the A. nidulans enzyme leads to a mutant with 5.8% of the recombinant wild-type carboxylase activity. All other mutations at this position resulted in unassembled proteins. Purified H295A and H324Q enzymes had elevated Km(RuBP) values and unchanged CO2/O2 specificity factors compared to recombinant wild-type.Abbreviations CABP D-2-carboxyarabinitol 1,5 bisphosphate - IPTG isopropyl-b-d-thiogalactopyranoside - L large subunit of rubisco - PAGE polyacrylamide gel electrophoresis - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-P2, ribulose 1,5 bisphosphate - S small subunit of rubisco - SDS sodium dodecyl sulfate - X-gal 5-bromo-4-chloro-3-indolyl-b-d-galactoside  相似文献   

10.
The quaternary structure of ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) from Rhodospirillum rubrum, an enzyme consisting of two large subunits, L2, was investigated by small-angle X-ray scattering. In the presence of HCO 3 - and Mg2+, rubisco is in the active state and displays a radius of gyration of 2.96 nm, a maximum diameter of 9.5 nm and a volume of 170 nm3. A model is presented where the subunits are arranged back-to-back, rotated relative to each other by 90°, and shifted by 1.3 nm. Upon inactivation by removal of HCO 3 - and Mg2+, the model swells slightly without any distinct changes in configuration. This contrasts with our previous observations with rubisco from Alcaligenes eutrophus, an enzyme composed of small (S) and large (L) subunits, L8S8, where inactivation gives rise to substantial changes in configuration.Abbreviations RuBP Ribulose-1,5-bisphosphate - 3-PGA 3-phosphoglyceric acid  相似文献   

11.
The exchange properties of the activator CO2 of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO2 on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO2. The exchange kinetics of the activator CO2 were monitored by activation of the enzyme to steady state in the presence of 12CO2, followed by addition of 14CO2 and determination of the amount of labeled CO2 trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (Kobs) for exchange with both the purified enzyme (0.45 min−1) and in illuminated chloroplasts (0.18 min−1) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO2 was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.  相似文献   

12.
D-ribulose-1,5-bisphosphate carboxylases purified from barley or formate-grown Pseudomonas oxalaticus were inactivated by 2,3-butadione. Pseudo first-order inactivation depended on the presence of borate and was reduced by product 3-phosphoglycerate. The half-times at 30°C and pH 8.3 in the presence of 2 mM 2,3-butadione are 10 and 60 minutes for the enzymes from P. oxalaticus and barley, respectively. Saturation kinetics and arginine modification were demonstrated for the enzyme from P. oxalaticus.  相似文献   

13.
The β and γ subunits of the Fo·Fl-ATP synthase complex of Rhodospirillum, rubrum chromatophores were removed in two consecutive steps. The resulting depleted chromatophores lost all their ATP synthesizing activity but retained 70% of the light-induced proton uptake. ATP synthesis could be restored by reattachment of the isolated β and γ subunits together, but not of either one of them separately. These data suggest that the γ and β subunits are required for the operation of the chromatophore ATP synthase, but do not seem to participate in the light-induced proton uptake.  相似文献   

14.
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.  相似文献   

15.
In vitro mutagenic techniques have generated an asp→glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C→A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn2+.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn2+.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site.  相似文献   

16.
Ribulose 1,5-bisphosphate carboxylase (EC.4.1.1.39) has been obtained from Nicotiana tabacum leaf homogenates with specific activites from 0.5 to 0.8 µmol CO2 fixed (mg protein min)-1. These activities are reconciled with much lower, previously reported activities. The results suggest that if the tobacco enzyme is assayed under optimum conditions there is little difference in the intrinsic specific activities of tobacco and spinach ribulose 1,5-bisphosphate carboxylase. Several factors affecting activity measurements were examined.  相似文献   

17.
Crystalline ribulose 1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) was isolated from tobacco (Nicotiana tabacum L.) leaf homogenates and analyzed for several characteristic oxygenase prosthetic groups. Analyses by atomic absorption and emission spectroscopy and neutron activation indicated that the crystalline protein contains less than 0.2 g-atoms of tightly bound copper or iron per mole (550,000 g) of enzyme. In addition, the absorption and fluorescence spectra of concentrated solutions of the crystalline protein gave no indication of the presence of a flavin nucleotide. Thus, the enzymatic oxygenation of ribulose 1,5-bisphosphate to yield P-glycolate, which is believed to comprise the initial reaction in the photorespiratory metabolism of higher plants, appears not to involve these cofactors in the catalytic mechanism.  相似文献   

18.
Yeoh HH  Badger MR  Watson L 《Plant physiology》1980,66(6):1110-1112
A survey of the Km(CO2) values of ribulose-1,5-bisphosphate carboxylase from 60 grass species shows that enzyme from C3 grasses consistently exhibits lower Km(CO2) than does that from C4 grasses. Systematically ordered variation in Km(CO2) of ribulose-1,5-bisphosphate carboxylases from C3 and C4 grasses is also apparent and, among C4 grasses, this shows some correlation with C4 types.  相似文献   

19.
The energy-linked membrane-bound inorganic pyrophosphatase of Rhodospirollumrubrum, G-9, has been solubilized with good yield from chromatophores using cholate in the presence of MgCl2. The enzyme has been partially purified using ammonium sulfate fractionation and gel chromatography. After fractionation the enzyme requires phospholipid for activity. The solubilized enzyme is specific for PPi and requires Mg2+ for activity as has been found for other PPiases.  相似文献   

20.
Two new nucleotides have been found in the formic acid extracts of Escherichiacoli, Clostridiumbotulinum, Bacillussubtilis and Rhodospirillumrubrum isolated during log phase growth. In E.coli the compounds are present at all times during cell growth but increase in amount during interruption of aeration and transition to stationary phase. They migrate close to ppGpp during one dimensional chromatography on PEI cellulose but are clearly separated from ppGpp by paper chromatography. The compounds are unstable on PEI cellulose and purification was effected by chromatography on A25 Sephadex ion exchange columns. Preliminary characterization indicates that the predominant compound is a dinucleoside polyphosphate and that both compounds contain a modified adenosine nucleoside.  相似文献   

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