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     

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-.     

A simplified purification and some properties of ribulose 1,5-diphosphate carboxylase from barley

A rapid procedure was developed for purifying ribulose 1,5-diphosphate carboxylase from barley leaves. After (NH₄)₂SO₄ fractionation, the unique sedimentation properties of the enzyme were exploited to effect a single step purification to 90% homogeneity. High speed centrifugation pelleted the enzyme with complete recovery of activity. Residual impurities were then removed by diethylaminoethyl cellulose chromatography and density gradient centrifugation. The purified protein exhibited size heterogeneity due to polymerization. The polymerization products were enzymatically active aggregates of ribulose 1,5-diphosphate carboxylase and were precipitated by an antibody specific for the enzyme.     

Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16.

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.     

The structure of form I crystals of D-ribulose-1,5-diphosphate carboxylase.

Single crystals of d-ribulose-1,5-diphosphate carboxylase from tobacco leaves, Nicotiana tabacum (variety Turkish Samsun), have been examined by X-ray diffraction, electron microscopy, and optical diffraction. Twelve molecules are loosely packed into a body-centered cubic unit cell, space group I4¹32 with cell dimension $\text{a = 383}\text{A}\text{̊}$. The asymmetric unit is one quarter of a molecule, and the minimum molecular symmetry is 222. This symmetry when combined with estimates of the two subunit masses and stoichiometry is compatible with a molecular structure of the composition L₈S₈ (L is large subunit, S is small). If all bonds between large and small subunits are equivalent, the true molecular symmetry is 422; this symmetry is consistent with molecular images in micrographs.     

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 and properties of chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from Alcaligenes eutrophus.

Chorismate mutase and prephenate dehydratase from Alcaligenes autophus H16 were purified 470-fold with a yield of 24%. During the course of purification, including chromatography on diethylaminoethyl (DEAE)-cellulose, phenylalanine-substituted Sepharose, Sephadex G-200 and hydrogyapatite, both enzymes appeared in association. The ratio of their specific activities remained almost constant. The molecular weight of chorismate mutase-prephenast dehydratase varied from 144,000 to 187,000 due to the three different determination methods used. Treatment of electrophoretically homogeneous mutase-dehydratase with sodium dodecyl sulfate dissociated the enzyme into a single component of molecular weight 47,000, indicating a tetramer of identical subunits. The isoelectric point of the bifunctional enzyme was 5.8. Prephenate dehydrogenase was not associated with other enzyme activities; it was separated from mutasedehydratase by DEAE-cellulose chromatgraphy. Chromatography on DEAE Sephadex, Sephadex G-200, and hydroxyapatite resulted in a 740-fold purification with a yield of 10%. The molecular weight of the enzyme was 55,000 as determined by sucrose gradient centrifugation and 65,000 as determined by gel filtration or electrophoresis. Its isoelectric point was pH 6.6. In the overall conversion of chorismate to phenylpyruvate, free prephenate was formed which accumulated in the reaction mixture. The dissociation of prephenate allowed prephenate dehydrogenase to compete with prephenate dehydratase for the substrate.     

N-Hydroxides, aminotriazole, and cyanide activate ribulosebisphosphate carboxylase formation in Alcaligenes eutrophus

Abstract The pole of the Gram-positive rod Bacillus subtilis is formed by the construction of a crosswall which is then split. The newly exteriorized wall comes under stress and stretches to form the developing pole. A model is proposed to account for the even bisection of the septum. It is based on an extension of our previous finding that autolysin action on living cells is increased when the protonmotive force is dissipated in any of a number of ways. The first site of enzymatic attack is that region of the peripheral wall that has become farther removed from the cytoplasmic membrane as the result of the envagination of the developing septum. Later, enzymatic action lead to the cleavage midway between the portions of cytoplasmic membrane delimiting the septum as this region is farthest removed from the source of protonmotive force.     

Fluoride, hydrogen, and formate activate ribulosebisphosphate carboxylase formation in Alcaligenes eutrophus

Alcaligenes eutrophus formed ribulosebisphosphate carboxylase (RuBPCase; EC 4.1.1.39) when grown on fructose. Addition of sodium fluoride (NaF) to fructose minimal medium resulted in a slightly decreased growth rate and a rapid fivefold increase in RuBPCase specific activity. With citrate, a glucogenic carbon source, RuBPCase was also formed, However, addition of NaF to cells growing on citrate resulted in a 50% decrease in RuBPCase specific activity. Among the enzymes of fructose catabolism, NaF (10 mM) inhibited enolase in vitro by 98% and gluconate 6-phosphate dehydratase by 87%. Inhibition of the dehydratase by NaF was insignificant in vivo, as determined with a mutant defective in phosphoglycerate mutase activity. Growth of this mutant on fructose was not inhibited by NaF, and only a minor increase in RuBPCase activity was observed. From these results, we concluded that the product of the enolase reaction, phosphoenolpyruvate, played a role in RuBPCase formation. Addition of H2 or formate to the wild type growing on fructose or citrate did not affect the growth rate but resulted in rapid formation of RuBPCase activity. Mutants impaired in H2 metabolism formed RuBPCase at a low rate during growth on fructose plus H2 but at a high rate on formate. Apparently, additional reductant from H2 or formate metabolism induced RuBPCase formation in A. eutrophus.     

Identification of two binding sites of the D-ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach for D-ribulose 1,5-bisphosphate and effectors of the carboxylation reaction.

Fluorimetric studies of the binding of d-ribulose 1,5-bisphosphate (RuP₂) and the effectors 6-phosphogluconate and fructose 1,6-bisphosphate to the d-ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach were correlated with the functions of these sugar phosphates in the carboxylation reaction. These agents compete for two binding sites of the enzyme. At relatively low concentrations they bind to an allosteric site, where 6-phosphogluconate and fructose 1,6-bisphosphate display their stimulating effect on the fixation of CO₂. At higher concentrations these compounds inhibit the carboxylation reaction and compete with RuP₂ for the reaction center of the carboxylase. Preincubation of the enzyme with low concentrations of RuP₂ (0.1–5 μm) inhibits the activity of these effectors as well as the effector-induced fluorescence changes of the enzyme-2-p-toluidinonapthalene-6-sulfonate (TNS) complex by competition for the regulatory center which could be identified as the high affinity binding site of the enzyme for RuP₂ with a K_D = 0.6 μm. The deactivation of the carboxylase which is observed on preincubation of the enzyme with RuP₂ in the absence of bicarbonate and Mg²⁺ cannot be correlated to the binding of RuP₂ to the effector site. The deactivation process occurs in an RuP₂ concentration range similar to that for CO₂ fixation.