Studies on a conformational change of tobacco ribulose diphosphate carboxylase induced by D-ribulose-1,5-diphosphate

In the presence of d-ribulose diphosphate, crystalline ribulose diphosphate carboxylase from Nicotiana tabacum leaves undergoes a profound change in solubility. The solubility change did not involve a Conformational change in molecular volume exceeding 2% as measured by sedimentation velocity suggesting no gross change in quaternary structure. However, the change in solubility did involve a tertiary structural change wherein some previously buried tyrosyl and tryptophyl residues became exposed, as indicated by difference spectrophotometry. Although the enzyme molecule has 8 binding sites for ribulose diphosphate, the Conformational change is complete after 4 substrate molecules are bound. A cooperative action among the subunits is proposed.     

Quaternary structure and oxygenase activity of D-ribulose-1,5-bisphosphate carboxylase from Hydrogenomonas eutropha.

Electrophoretically homogeneous ribulose-1,5-bisphosphate (RuBP) carboxylase was obtained from autotropically grown Hydrogenomonas eutropha by sedimentation of the 105,000 X g supernatant in a discontinuous sucrose gradient and by ammonium sulfate fractionation followed by another sucrose gradient centrifugation. The molecular weight of the enzyme determined by light scattering was 490,000 +/- 15,000. The enzyme could be dissociated by sodium dodecyl sulfate into three types of subunits, and the molecular weights (+/- 10%) could be measured. There were two species of large subunits, L and L' (molecular weight 56,000 and 52,000, respectively) and one species of small subunits (molecular weight, 15,000). The mole ratio of L to L' was 5:3, and the overall mole ratio of the small to large subunits was 1.08. The simplest quaternary structure of the enzyme is L5L'3S8. The enzyme contained RuBP oxygenase activity as evidenced by the O2-dependent production of phosphoglycolate and 3-phosphoglyceric acid in equimolar quantities from RuBP.     

Bicarbonate stabilization of ribulose 1,5-diphosphate carboxylase.

The carboxylase and oxygenase activities of purified soybean ribulose 1,5-di-P carboxylase (EC4.1.1.39) were unstable when reactions were initiated with enzyme. Time courses of carboxylase and oxygenase activities were curvilinear, approximating hyperbolas. Double reciprocal plots of amount of CO2 incorporated and P-glycolate produced vs. time were constructed to determine a constant representing the half-time of initial enzyme activity, K. K increased with increasing bicarbonate concentration but was independent of O2 tensions between 0.21 and 5 atm. When time courses of carboxylase and oxygenase activities were determined simultaneously, K was identical for both activities. Linear time courses were obtained py preincubation of the enzyme for 10 min in the absence of bicarbonate or by adding 46 mM MgCl2 to the reaction mixture. The observed bicarbonate-dependent decline in ribulose 1,5-di-P carboxylase activity with time is the probable cause for the anomalously high Km(CO2) values previously reported for this enzyme. In the experiments reported here, the apparent Km(CO2) at pH 8.5 increased from 6 muM CO2 at zero time to 78 muM CO2 at 10 min. The corresponding bicarbonate Km values ar 1;3 and 17 mM, respectively, The interaction between bicarbonate and enzyme may be important in the light activation of photosynthetic CO2 fixation in vivo.     

Different molecular forms of D-ribulose-1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides.

Ribulose-1,5-bisphosphate (Rbu-P2) carboxylase isolated from Rhodopseudomonas sphaeroides 2.4.1.Ga was separated into two different forms by DEAE-cellulose column chromatography. Both forms, designated Peak I and Peak II have been purified to homogeneity by the criterion of polyacrylamide disc-gel electrophoresis. The Peak I carboxylase has a molecular weight of 550,000, while the Peak II carboxylase is a smaller protein having a molecular weight of approximately 360,000. Sodium dodecyl sulfate electrophoresis revealed a large subunit for both enzymes which migrates similarly to the large subunit of spinach Rbu-P2 carboxylase. The Peak I enzyme also exhibited a small subunit having a molecular weight of 11,000. No evidence for a smaller polypeptide was found associated with the Peak II enzyme. Antisera prepared against the Peak I enzyme inhibited Peak I enzymatic activity, but had no effect on the activity of the Peak II enzyme. The two enzymes exhibited marked differences in catalytic properties. The Peak I enzyme exhibits optimal activity at pH 8.0 and is inhibited by low concentrations of 6-phosphogluconate, while the Peak II enzyme has a pH optimum of 7.2 and is relatively insensitive to 6-phosphogluconate.     

Ribulose 1,5-diphosphate carboxylase and Chlorobium thiosulfatophilum

1. Cell-free extracts of the photosynthetic bacterium Chlorobium thiosulfatophilum, strains 8327 and Tassajara, were assayed for ribulose 1,5-diphosphate (RuDP) carboxylase and phosphoribulokinase-the two enzymes peculiar to the reductive pentose phosphate cycle. 2. RuDP carboxylase was consistently absent in strain 8327. The Tassajara strain showed a low RuDP-dependent CO₂ fixation activity that was somewhat higher in cells following transatlantic air shipment than in freshly grown cells. The stability and behaviour of this activity in sucrose density gradients were similar to those described by other workers. 3. The radioactive carboxylation products formed in the presence of RuDP by enzyme preparations from the Tassajara strain did not include 3-phosphoglycerate-the known product of the RuDP carboxylase reaction, but instead consisted of the unrelated acids glutamate, aspartate and malate. 4. Phosphoribulokinase was absent in all preparations of the two Chlorobium strains tested. By contrast, phosphoribulokinase as well as RuDP carboxylase were readily demonstrated in preparations from pea chloroplasts and the photosynthetic bacterium Rhodospirillum rubrum. 5. It is concluded that C. thiosulfatophilum appears to lack RuDP carboxylase, phosphoribulokinase, and hence, the reductive pentose phosphate cycle.Support of a J. S. Guggenheim Fellowship is gratefully acknowledged     

Purification, quaternary structure, composition, and properties of D-ribulose-1,5-bisphosphate carboxylase from Thiobacillus intermedius.

D-Ribulose-1,5-bisphosphate (RuBP) carboxylase has been purified from glutamate-CO2-S2O3(2)-grown Thiobacillus intermedius by pelleting the enzyme from the high-speed supernatant and by intermediary crystallization followed by sedimentation into a discontinuous 0.2 to 0.8 M sucrose gradient. The enzyme was homogeneous by the criteria of electrophoresis on polyacrylamide gels of several acrylamide concentrations, sedimentation velocity and equilibrium measurements, and electron microscopic observations of negatively stained preparations. The molecular weights of the enzyme determined by sedimentation equilibrium and light-scattering measurements averaged 462,500 +/- 13,000. The enzyme consisted of closely similar or identical polypeptide chains of a molecular weight of 54,500 +/- 5,450 determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The S(0)20,w of the enzyme was 18.07S +/- 0.22. Electron microscopic examination suggested that the octomeric enzyme (inferred from the molecular measurements mentioned) had a cubical structure. The specific activity of the enzyme was 2.76 mumol of RuBP-dependent CO2 fixed/min per mg of protein (at pH 8 and 30 C), and the turnover number in terms of moles of CO2 fixed per mole of catalytic site per second was 2.6. The enzyme was stable for 3 months at -20 C and at least 4 weeks at 0 C. The apparent Km for CO2 was 0.75 mM, and Km values for RuBP and Mg2+ were 0.076 and 3.6 mM, respectively. Dialyzed enzyme could be fully reactivated by the addition of 20 mM Mg2+ and partially reactivated by 20 mM Co2+, but Cd2+, Mn2+, Ca2+, and Zn2+ had no effect. The compound 6-phosphogluconate was a linear competitive inhibitor with respect to RuBP when it had been preincubated with enzyme, Mg2+, and HCO3-.     

Isolation and characterization of wheat ribulose-1,5-diphosphate carboxylase

Wheat ribulose-1,5-diphosphate carboxylase purified to homogeneity had a MW of 540 000, sedimentation coefficient (S_{20, W}) of 18.5 S, apparent diffusion constant (D_app) 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 (S_{20, W} = 3.0 S) and 13 500 (S_{20, 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.     

Inhibition of ribulose 1,5-diphosphate carboxylase by 6-phosphogluconate

6-Phosphogluconate is a much more effective inhibitor of the photosynthetic carboxylation enzyme, ribulose-1, 5-diphosphate carboxylase, than other sugar phosphates and sugar acids of the reductive and oxidative pentose phosphate cycles. The inhibition appears to be noncompetitive with ribulose 1,5-diphosphate. Since 6-phosphogluconate is unique to the oxidative cycle and inhibits at concentrations comparable to those found in vivo, it is proposed that its inhibition of the carboxylase may be a regulatory factor. If so, it would operate during darkness as a different control factor from those factors postulated to activate the carboxylase during photosynthesis.     

Purification,some properties and quaternary structure of thed-ribulose 1,5-diphosphate carboxylase ofAlcaligenes eutrophus

d-Ribulose 1,5-diphosphate carboxylase has been purified from autotrophically grown cells of the facultative chemolithotrophic hydrogen bacteriumAlcaligenes eutrophus. The enzyme was homogeneous by the criteria of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 505000 determined by gel filtration and sucrose density gradient centrifugation, and a sedimentation coefficient of 18.2 S was obtained. It was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis that the enzyme consists of two types of subunits of molecular weight 52000 and 13000.Electron microscopy on the intact and the partially dissociated enzyme lead to the construction of a model for the quaternary structure of the enzyme which is composed of 8 large and 8 small subunits. The most probable symmetry of the enzyme molecule is 4:2:2.Michaelis constant (K _m ) values for ribulose 1,5-diphosphate, Mg^2-, and CO₂ were 0.59 mM, 0.33 mM, and 0.066 mM measured under air. Oxygen was a competitive inhibitor with respect to CO₂ suggesting that the enzyme also exhibits an oxygenase activity. The oxygenolytic cleavage of ribulose 1,5-diphosphate was shown and a 1:1 stoichiometry between oxygen consumption and 3-phosphoglycerate formation observed.Abbreviations DTE dithioerythritol - EDTA ethylenediamine tetraacetate - RuDP d-ribulose 1,5-diphosphate