Ribulose 1,5-bisphosphate and activation of the carboxylase in the chloroplast

Ribulose 1,5-bisphosphate in the chloroplast has been suggested to regulate the activity of the ribulose bisphosphate carboxylase/oxygenase. To generate high levels of ribulose bisphosphate, isolated and intact spinach chloroplasts were illuminated in the absence of CO₂. Under these conditions, chloroplasts generate internally up to 300 nanomoles ribulose 1,5-bisphosphate per milligram chlorophyll if O₂ is also absent. This is equivalent to 12 millimolar ribulose bisphosphate, while the enzyme, ribulose bisphosphate carboxylase, offers up to 3.0 millimolar binding sites for the bisphosphate in the chloroplast stroma. During illumination, the ribulose bisphosphate carboxylase is deactivated, due mostly to the absence of CO₂ required for activation. The rate of deactivation of the ribulose bisphosphate carboxylase was not affected by the chloroplast ribulose bisphosphate levels. Upon addition of CO₂, the carboxylase in the chloroplast was completely reactivated. Of interest, addition of 3-phosphoglycerate stopped deactivation of the carboxylase in the chloroplast while ribulose bisphosphate accumulated. With intact chloroplasts in light, no correlation between deactivation of the carboxylase and ribulose bisphosphate levels could be shown.     

Ribulose 1,5-bisphosphate carboxylase. Effect on the catalytic properties of changing methionine-330 to leucine in the Rhodospirillum rubrum enzyme.

Oligonucleotide-directed mutagenesis of cloned Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase with a synthetic 13mer oligonucleotide primer was used to effect a change at Met-330 to Leu-330. The resultant enzyme was kinetically examined in some detail and the following changes were found. The Km(CO2) increased from 0.16 to 2.35 mM, the Km(ribulose bisphosphate) increased from 0.05 to 1.40 mM for the carboxylase reaction and by a similar amount for the oxygenase reaction. The Ki(O2) increased from 0.17 to 6.00 mM, but the ratio of carboxylase activity to oxygenase activity was scarcely affected by the change in amino acid. The binding of the transition state analogue 2-carboxyribitol 1,5-bisphosphate was reversible in the mutant and essentially irreversible in the wild type enzyme. Inhibition by fructose bisphosphate, competitive with ribulose bisphosphate, was slightly increased in the mutant enzyme. These data suggest that the change of the residue from methionine to leucine decreases the stability of the enediol reaction intermediate.     

Influence of Inorganic Phosphate on Photosynthesis of Wheat Chloroplasts: II. RIBULOSE BISPHOSPHATE CARBOXYLASE ACTIVITY

Isolated wheat chloroplasts were pre-incubated in the dark inthe presence of various concentrations of inorganic phosphatewith or without carbon dioxide, oxaloacetate, glycerate, and3-phosphoglycerate. The effect of subsequent illumination onphotosynthetic oxygen evolution, ribulose bisphosphate carboxylaseactivity, ATP content, and ribulose bisphosphate content wasinvestigated. Inorganic phosphate had little effect on ribulosebisphosphate carboxylase activity in darkness or during theinitial phase of illumination, but it prevented the declinein activity that occurred during later stages of illumination,when photoreduction of CO₂ was decreasing in rate. Additionof inorganic phosphate to chloroplasts illuminated without phosphaterestored the ribulose bisphosphate carboxylase activity, increasedthe ATP, and decreased the ribulose bisphosphate in the organelles.The responses to CO₂, oxaloacetate, glycerate, and 3-phosphoglyceratesuggest that the decreased activity of ribulose bisphosphatecarboxylase during photosynthesis results from ATP consumption. Purified ribulose bisphosphate carboxylase was activated byinorganic phosphate, but this activation did not occur in thepresence of ATP. ATP inhibited ribulose bisphosphate carboxylasewhen it was present in combination with various photosyntheticmetabolites. Inactivation of ribulose bisphosphate carboxylase in chloroplasts,illuminated in the absence of inorganic phosphate, is not dueto lack of activation by inorganic phosphate or ATP. It mayresult from decreased stromal pH. Key words: Ribulose bisphosphate carboxylase, Chloroplasts, Wheat, Phosphate, ATP     

Photosynthesis and ribulose 1,5-bisphosphate levels in intact chloroplasts

The response of ribulose 1,5-bisphosphate levels and CO(2) fixation rates in isolated, intact spinach chloroplasts to pyrophosphate, triose phosphates, dl-glyceraldehyde, O(2), catalase, and irradiance during photosynthesis has been studied. Within 1 minute in the light, a rapid accumulation of ribulose bisphosphate was measured in most preparations of intact chloroplasts, and this subsequently dropped as CO(2) fixation increased. Pyrophosphate, triose phosphates, and catalase increased CO(2) fixation and also the levels of ribulose bisphosphate. CO(2) fixation was inhibited by dl-glyceraldehyde and O(2) with corresponding decreases in ribulose bisphosphate. When the rate of photosynthesis decreased at limiting irradiances (low light), the level of ribulose bisphosphate in the chloroplast did not always decrease, suggesting that ribulose bisphosphate was not limiting CO(2) fixation under these conditions. When triose phosphates (fructose bisphosphate plus aldolase) were added to suspensions of chloroplasts at low irradiances, ribulose bisphosphate increased while CO(2) fixation decreased. These observations provide considerable evidence that high ribulose bisphosphate levels clearly are not solely sufficient to permit rapid rates of CO(2) fixation, but that factors other than ribulose bisphosphate concentration are overriding the control of photosynthesis.Isolated chloroplasts are capable of using carbon reserves to produce considerable ribulose bisphosphate. Upon illumination in the absence of CO(2) and O(2), intact chloroplasts produced up to 13 millimolar ribulose bisphosphate.     

2-Bromoacetylaminopentitol 1,5-bisphosphate as an affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

2-Bromoacetylaminopentitol 1,5-bisphosphate (BrAcNH-pentitol-P2) (an epimeric mixture of 2-bromoacetylamino-2-deoxy-D-ribitol bisphosphate and 2-bromoacetylamino-2-deoxy-D-arabinitol 1,5-bisphosphate) has been synthesized from D-ribulose 1,5-bisphosphate by reductive amination with sodium cyanoborohydride followed by bromoacetylation of the resultant amine with bromoacetyl bromide. Under conditions that favor full activation of the enzyme, ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is completely inactivated by BrAcNH-pentitol-P2 in a pseudo-first order process. A rate saturation is observed with a minimal inactivation half-life of 38 min and Kinact for reagent of 0.38 mM. The competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduces the rate of inactivation, and kinetic analyses are consistent with the protection reflecting true competition of inhibitor and reagent for the same site. As shown with isotopically labeled reagent, complete inactivation is associated with covalent incorporation of 1.1 mol of reagent/mol of subunit. Based on reversibility of inactivation by thiolysis and based on analysis of labeled products in acid hydrolysates of the modified enzyme, a methionyl sulfonium salt is the reaction product. In the absence of CO2 and Mg2+ (ligands required for activation), the enzyme is resistant to BrAcNH-pentitol-P2, which suggests that the site-specific modification of a methionyl residue requires a fully functional catalytic center.     

A model for the kinetics of activation and catalysis of ribulose 1,5-bisphosphate carboxylase.

Further evidence for time-dependent interconversions between active and inactive states of ribulose 1,5-bisphosphate carboxylase is presented. It was found that ribulose bisphosphate oxygenase and ribulose bisphosphate carboxylase could be totally inactivated by excluding CO2 and Mg2+ during dialysis of the enzyme at 4 degrees C. When initially inactive enzyme was assayed, the rate of reaction continually increased with time, and the rate was inversely related to the ribulose bisphosphare concentration. The initial rate of fully activated enzyme showed normal Michaelis-Menten kinetics with respect to ribulose bisphosphate (Km = 10muM). Activation was shown to depend on both CO2 and Mg2+ concentrations, with equilibrium constants for activation of about 100muM and 1 mM respectively. In contrast with activation, catalysis appeared to be independent of Mg2+ concentration, but dependent on CO2 concentration, with a Km(CO2) of about 10muM. By studying activation and de-activation of ribulose bisphosphate carboxylase as a function of CO2 and Mg2+ concentrations, the values of the kinetic constants for these actions have been determined. We propose a model for activation and catalysis of ribulose bisphosphate carboxylase: (see book) where E represents free inactive enzyme; complex in parentheses, activated enzyme; R, ribulose bisphosphate; M, Mg2+; C, CO2; P, the product. We propose that ribulose bisphosphate can bind to both the active and inactive forms of the enzyme, and slow inter-conversion between the two states occurs.     

The biosynthesis of ribulose bisphosphate carboxylase. Uncoupling of the synthesis of the large and small subunits in isolated soybean leaf cells

Isolated leaf cells from soybean (Glycine max) incorporate [35S]methionine into protein at a linear rate for at least 5h. Analysis of the products of incorporation by one-dimensional and two-dimensional polyacrylamide gel electrophoresis shows that major products are the large and small subunits of the chloroplast enzyme, ribulose bisphosphate carboxylase. The large subunit is synthesized by chloroplast ribosomes and the small subunit by cytoplasmic ribosomes. Addition of chloramphenicol to the cells reduces incorporation into the large subunit without affecting incorporation into the products of cytoplasmic ribosomes. Addition of cycloheximide or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide stops incorporation into the small subunit, but large subunit continues to be made for at least 4 h. For accurate estimates of incorporation into the large subunit, it is essential to use two-dimensional gel electrophoresis, because the large subunit region on one-dimensional gels is contaminated with the products of cytoplasmic ribosomes. Newly synthesized large subunits continue to enter complete molecules of ribulose bisphosphate carboxylase in the absence of small subunit synthesis. These results suggest that, in contrast to the situation in algal cells, the synthesis of the two subunits of ribulose bisphosphate carboxylase in the different subcellular compartments of higher plant cells is not tightly coupled over short time periods, and that a pool of small subunits exists in these cells. The results are disucssed in relation to possible mechanisms for the integration of the synthesis of the large and small subunits of ribulose bisphosphate carboxylase.     

Photorespiration stoichiometry in leaves estimated by combined physical and stereochemical methods: allowance for isomerase-catalyzed 3H losses in ribulose bisphosphate regeneration

We showed previously [K.R. Hanson and R.B. Peterson (1986) Arch. Biochem. Biophys. 246, 332-346] that under steady-state photosynthetic conditions the fraction of ribulose bisphosphate oxidized and the fraction of glycolate carbon photorespired (the stoichiometry of photorespiration) may be estimated in leaves by a combination of physical and stereochemical methods. The calculations assumed that when (3R)-D-[3-3H1,3-14C]glyceric acid is supplied to illuminated leaf discs the only loss of 3H from the combined photosynthetic and photorespiratory system is the result of glycolate oxidase action; i.e., the isomerase-catalyzed losses in the regeneration of ribulose bisphosphate are negligible. The present study of tobacco leaf discs under zero-photorespiration conditions (low O2 and high CO2 concentrations), and also of maize leaf discs, shows that some 3H losses occur (between 8 and 13% of the 3H at C-1 of ribulose 5-phosphate). The calculated loss varied moderately with temperature but did not vary when the flux of ribulose bisphosphate formation was altered by changing the irradiance. The calculated loss under zero-photorespiration conditions, therefore, may be used to calculate ribulose bisphosphate and glycolate partitioning under other conditions. Earlier experiments on the influence of O2 and CO2 concentrations of temperature on the partitioning of ribulose bisphosphate and glycolate have been reexamined. The loss corrections decreased all values for the fraction of ribulose bisphosphate oxidized and increased all values for the stoichiometry of photorespiration. Essentially all stoichiometry values were above the theoretical lower limit of 25%. The previous conclusion that the stoichiometry of photorespiration substantially exceeds 25% at higher O2 concentrations and higher temperatures is unchanged. The results with maize leaf discs implied that there is very little oxidation of ribulose 1,5-bisphosphate under normal-air conditions; i.e., photorespiration is indeed suppressed, not merely hidden, by efficient refixation of CO2.     

A calorimetric study on calcium binding by troponin C from bullfrog skeletal muscle

Microcalorimetric titrations of bullfrog (Rana catesbeiana) skeletal troponin C with Ca2+ were carried out in the absence of Mg2+ at 25 degrees C and at pH 7.0. The observed enthalpy titration curve was divided into three stages. The first stage of the titration (up to 2 mol of Ca2+/mol of protein) was characterized as an extremely exothermic process (delta H = -52 kJ/mol of site), the second one (titration from 2 to 3 mol of Ca2+/mol of protein) as a weakly endothermic process (delta H = +26 kJ/mol of site), and the final one (over 3 mol of Ca2+/mol of protein) as a moderately exothermic process (delta H = -35 kJ/mol of site). The endothermic process of Ca2+ binding to the third site (the second stage) has the same property as that of the Ca2+ binding to every site of calmodulin but is distinctly different from those of the calmodulin-trifluoperazine complex and parvalbumins. This may suggest that an endothermic nature of Ca2+ binding, the reaction being driven solely by entropy change, is characteristic of the regulatory reactions of Ca2+ binding proteins accompanying the interaction with other proteins. The third Ca2+ binding site of bullfrog troponin C is, therefore, possibly involved in the regulation of muscle contraction.     

Modification of Rhodospirillum rubrum ribulose bisphosphate carboxylase with pyridoxal phosphate. 1. Identification of a lysyl residue at the active site.

Ribulose 1,5-bisphosphate carboxylase isolated from Rhodospirillum rubrum was strongly inhibited by low concentrations of pyridoxal 5'-phosphate. Activity was protected by the substrate ribulose bisphosphate and to a lesser extent by other phosphorylated compounds. Pyridoxal phosphate inhibition was enhanced in the presence of magnesium and bicarbonate, but not in the presence of either compound alone. Concomitant with inhibition of enzyme activity, pyridoxal phosphate forms a Schiff base with the enzyme which is reversible upon dialysis and reducible with sodium borohydride. Subsequent to reduction of the Schiff base with tritiated sodium borohydride, tritiated N6-pyridoxyllysine could be identified in the acid hydrolysate of the enzyme. Only small amounts of this compound were present when the reduction was performed in the presence of carboxyribitol bisphosphate, an analogue of the intermediate formed during the carboxylation reaction. Therefore, it is concluded that pyridoxal phosphate modifies a lysyl residue close to or at the active site of ribulose bisphosphate carboxylase.     

The binding of copper ions to copper-free bovine superoxide dismutase. Kinetic aspects.

The kinetics of reconstitution of bovine superoxide dismutase from Cu2+ and the copper-free enzyme have been studied by activity, u.v.-absorption, electron-paramagnetic-resonance and pulsed-nuclear-magnetic-resonance measurements. The process appears to be first-order up to 80% completion in most conditions, and is pH-dependent, with an apparent pK of 6.5. U.v.-absorption and solvent proton relaxation rate measurements show that fast binding of Cu2+ occurs, and the initial ligands are likely to be, at least in part, those of the native active site. The recovery of the native activity and spectroscopic properties is a slow process with activation energies of 92 kJ/mol at pH 5.3 and 8.4kJ/mol at pH 8.1 and can be described as a rearrangement of the site around the bound metal. The rate of this process is lower in partially recombined protein samples, probably because of intersubunit interactions.     

Examination of the function of active site lysine 329 of ribulose-bisphosphate carboxylase/oxygenase as revealed by the proton exchange reaction

Diverse approaches that include site-directed mutagenesis have indicated a catalytic role of Lys-329 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. To determine whether Lys-329 is required for the initial enolization of ribulose bisphosphate or for some subsequent step in the overall reaction pathway, the competence of position 329 mutant proteins (devoid of carboxylase activity) in catalyzing exchange of solvent protons with the C-3 proton of substrate has now been examined. Irrespective of the amino acid substitution for Lys-329, the mutant protein retains 2-6% of the wild-type activity in the proton exchange reaction. The complete stability of ribulose bisphosphate during the enolization catalyzed by mutant protein suggests that the major effect of Lys-329 is to facilitate the addition of gaseous substrates (CO2 or O2) to the enediol intermediate. The exchange reaction requires Mg2+, is CO2-dependent, and is inhibited by the transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate. A mutant protein in which Lys-191, the site for carbamylation by CO2 in an obligatory activation step, is replaced by a cysteinyl residue totally lacks proton exchange activity. Barely detectable exchange activity (approximately 0.2% of wild-type) is displayed by the Lys-166----Cys mutant protein, consistent with the previously implicated role of Lys-166 in the deprotonation of ribulose bisphosphate. Retention of exchange activity by the Glu-48----Gln mutant protein, which is slightly active in overall carboxylation, demonstrates that active site Glu-48, like Lys-329, exerts its major effect at some step subsequent to the initial enolization.     

Oxygen isotope effects on the ribulosebisphosphate oxygenase reaction

The oxygen isotope effect at the substrate O2 on the oxygenase reaction of ribulose bisphosphate carboxylase/oxygenase from spinach is pH and metal dependent. The pH dependence between pH 7.4 and 8.9 is different with Mg2+ (steady decrease in this isotope effect from 1.036 to 1.030) and Mn2+ (minimum isotope effect of 1.028 at pH 8.0). Deuteration of the substrate ([3-2H]ribulose bisphosphate) has no influence on the isotope effect. The results are interpreted as a direct participation of the metal ion in the oxygen-sensitive step, i.e. carbon-oxygen bond formation and the stabilization of the intermediates. In the overall reaction oxygen addition is a major rate-limiting step, and the observed isotope effect is probably close to the intrinsic oxygen isotope effect of the reaction. The basic mechanisms for carboxylation and oxygenation of ribulose bisphosphate appear to be the same.     

Presence of two subunit types in ribulose 1,5-bisphosphate carboxylase from Thiobacillus intermedius.

Ribulose bisphosphate carboxylase (EC 4.1.1.39) has been purified to homogeneity from glutamate-CO2-thiosulfate-grown Thiobacillus intermedius by pelleting the protein from the 93,000 X g supernatant fluid followed by ammonium sulfate fractionation and sedimentation into a discontinuous sucrose density gradient. The molecular weight of the native protein approximated that of the higher plant enzyme (550,000) based on its relative electrophoretic mobility in polyacrylamide disc gels compared with that of standards of known molecular weight, including crystalline tobacco ribulose bisphosphate carboxylase. Sodium dodecyl sulfate electrophoresis in 12% polyacrylamide disc gels and Sephadex G-100 chromatography in the presence of sodium dodecyl sulfate indicated that the purified Thiobacillus protein, like the tobacco enzyme, consisted of two types of nonidentical subunits. The molecular weights of the large and small subunits were estimated to be about 55,000 and 13,000, respectively, by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase activity of the protein purified from spinach leaves and T. intermedius responded similarly to the effectors reduced nicotinamide adenine dinucleotide phosphate and 6-phosphogluconate. Contrary to a previous report (K. Purohit, B. A. McFadden, and A. L. Cohen, J. Bacteriol. 127:505-515, 1976), these results indicate that ribulose bisphosphate carboxylase purified from Thiobacillus intermedius closely resembles the higher plant enzyme with respect to quaternary structure, molecular weight, and regulatory properties.     

Coordinate expression of hydrogenase and ribulose bisphosphate carboxylase in Rhizobium japonicum Hupc mutants.

In contrast to the wild type, H2 uptake-constitutive mutants of Rhizobium japonicum expressed both hydrogenase and ribulose bisphosphate carboxylase activities when grown heterotrophically. However, as bacteroids from soybean root nodules, the H2 uptake-constitutive mutants, like the wild type, did not express ribulose bisphosphate carboxylase activity.     

High temperature effects on RuBP carboxylase and carbonic anhydrase activity in two tomato cultivars

The effects of temperature on ribulose bisphosphate carboxylase activity were studied in two tomato ( Lycopersicon esculentum Mill.) cultivars which differed in sensitivity to high temperatures. The heat tolerant cultivar, Saladette, had a smaller reduction in photosynthesis and a smaller increase in mesophyll resistance then the sensitive cultivar Roma VF, after 24 h at 35 to 40°C. One hour in vitro treatments at 50°C decreased the activity of ribulose bisphosphate carboxylase extracted from Roma VF by 75%, while Saladette was not affected. Heat stress to the entire plant caused greater inhibition of ribulose bisphosphate carboxylase in the heat sensitive cultivar. Ribulose bisphosphate carboxylase activity in both cultivars decreased with heat treatment but recovered under normal temperatures. Ribulose bisphosphate oxygenase activity decreased similarly in both cultivars under 37/18°C day/night temperatures, which resulted in an apparent change in the relative carboxylase/oxygenase activity of the two cultivars. Carbonic anhydrase activity was slightly greater in Saladette than in Roma VF but no significant decrease in activity was observed in plants exposed to high temperatures.     

A kinetic study of ribulose bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum.

The activation kinetics of purified Rhodospirillum rubrum ribulose bisphosphate carboxylase were analysed. The equilibrium constant for activation by CO(2) was 600 micron and that for activation by Mg2+ was 90 micron, and the second-order activation constant for the reaction of CO(2) with inactive enzyme (k+1) was 0.25 X 10(-3)min-1 . micron-1. The latter value was considerably lower than the k+1 for higher-plant enzyme (7 X 10(-3)-10 X 10(-3)min-1 . micron-1). 6-Phosphogluconate had little effect on the active enzyme, and increased the extent of activation of inactive enzyme. Ribulose bisphosphate also increased the extent of activation and did not inhibit the rate of activation. This effect might have been mediated through a reaction product, 2-phosphoglycolic acid, which also stimulated the extent of activation of the enzyme. The active enzyme had a Km (CO2) of 300 micron-CO2, a Km (ribulose bisphosphate) of 11--18 micron-ribulose bisphosphate and a Vmax. of up to 3 mumol/min per mg of protein. These data are discussed in relation to the proposed model for activation and catalysis of ribulose bisphosphate carboxylase.     

The Regulation of Photosynthesis in Leaves of Field-Grown Spring Wheat (Triticum aestivum L., cv Albis) at Different Levels of Ozone in Ambient Air

Wheat (Triticum aestivum L. cv Albis) was grown in open-top chambers in the field and fumigated daily with charcoal-filtered air (0.015 microliters per liter O₃), nonfiltered air (0.03 microliters per liter O₃), and air enriched with either 0.07 or 0.10 microliters per liter ozone (seasonal 8 hour/day [9 am-5 pm] mean ozone concentration from June 1 until July 10, 1987). Photosynthetic ¹⁴CO₂ uptake was measured in situ. Net photosynthesis, dark respiration, and CO₂ compensation concentration at 2 and 21% O₂ were measured in the laboratory. Leaf segments were freeze-clamped in situ for the determination of the steady state levels of ribulose 1,5-bisphosphate, 3-phosphoglycerate, triose-phosphate, ATP, ADP, AMP, and activity of ribulose, 1,5-bisphosphate carboxylase/oxygenase. Photosynthesis of flag leaves was highest in filtered air and decreased in response to increasing mean ozone concentration. CO₂ compensation concentration and the ratio of dark respiration to net photosynthesis increased with ozone concentration. The decrease in photosynthesis was associated with a decrease in chlorophyll, soluble protein, ribulose bisphosphate carboxylase/oxygenase activity, ribulose bisphosphate, and adenylates. No decrease was found for triose-phosphate and 3-phosphoglycerate. The ratio of ATP to ADP and of triosephosphate to 3-phosphoglycerate were increased suggesting that photosynthesis was limited by pentose phosphate reductive cycle activity. No limitation occurred due to decreased access of CO₂ to photosynthetic cells since the decrease in stomatal conductance with increasing ozone concentration did not account for the decrease in photosynthesis. Ozonestressed leaves showed an increased degree of activation of ribulose bisphosphate carboxylase/oxygenase and a decreased ratio of ribulose bisphosphate to initial activity of ribulose bisphosphate carboxylase/oxygenase. Nevertheless, it is suggested that photosynthesis in ozone stressed leaves is limited by ribulose bisphosphate carboxylation possibly due to an effect of ozone on the catalysis by ribulose bisphosphate carboxylase/oxygenase.     

Ribulose bisphosphate carboxylase/oxygenase in toluene-permeabilized Rhodospirillum rubrum.

Toluene-permeabilized Rhodospirillum rubrum cells were used to study activation of and catalysis by the dual-function enzyme ribulose bisphosphate carboxylase/oxygenase. Incubation with CO2 provided as HCO3-, followed by rapid removal of CO2 at 2 degrees C and subsequent incubation at 30 degrees C before assay, enabled a determination of decay rates of the carboxylase and the oxygenase. Half-times at 30 degrees C with 20 mM-Mg2+ were 10.8 and 3.7 min respectively. Additionally, the concentrations of CO2 required for half-maximal activation were 56 and 72 microM for the oxygenase and the carboxylase respectively. After activation and CO2 removal, inactivation of ribulose bisphosphate oxygenase in the presence of 1 mM- or 20mM-Mn2+ was slower than that with the same concentrations of Co2+ or Mg2+. Only the addition of Mg2+ supported ribulose bisphosphate carboxylase activity, as Mn2+, Co2+ and Ni2+ had no effect. A pH increase after activation in the range 6.8-8.0 decreased the stability of the carboxylase but in the range 7.2-8.0 increased the stability of the oxygenase. With regard to catalysis. Km values for ribulose 1,5-bisphosphate4- were 1.5 and 67 microM for the oxygenase and the carboxylase respectively, and 125 microM for O2. Over a broad range of CO2 concentrations in the activation mixture, the pH optima were 7.8 and 8-9.2 for the carboxylase and the oxygenase respectively. The ratio of specific activities was constant (9:1 for the carboxylase/oxygenase) of ribulose bisphosphate carboxylase/oxygenase in toluene-treated Rsp. rubrum. Below concentrations of 10 microM-CO2 in the activation mixture, this ratio increased.     

Functional analysis of the putative catalytic bases His-321 and Ser-368 of Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase by site-directed mutagenesis.

Numerous candidates have been suggested according to chemical and structural criteria for the active site base of ribulose bisphosphate carboxylase/oxygenase that catalyzes substrate enolization. We evaluate the functional significance of two such candidates, His-321 and Ser-368 of the Rhodospirillum rubrum enzyme, by site-directed mutagenesis. Position 321 mutants retain 3-12% of wild-type rates of both overall carboxylation and the initial enolization, with little effect on Km for CO2 or ribulose bisphosphate. Position 368 mutants exhibit approximately 1% of wild-type carboxylation but 4-9% of enolization, also accompanied by little effect on Km values. The modest catalytic facilitations elicited by these residues are incompatible with either acting as the crucial base. The enhanced efficiency of the position 368 mutants in enolization versus carboxylation clearly indicates that Ser-368 effects catalysis preferentially beyond the point of proton abstraction. Both sets of mutants bind the reaction intermediate analogue, 2-carboxy-D-arabinitol bisphosphate, stoichiometrically. Ligand exchange from complexes with position 321 mutants is increased relative to wild type, whereas complexes with position 368 mutants are more exchange-inert. Therefore, His-321 may assist stabilization of the transition state mimicked by the analogue.