Effects of CO2, O2 and temperature on a high-affinity form of ribulose diphosphate carboxylase-oxygenase from spinach

A high-affinity form of ribulose diphosphate carboxylase, observed transiently in spinach-leaf extracts soon after extraction, was inhibited by O₂ competitively with respect to CO₂. Analogously, the ribulose diphosphate oxygenase activity for this form was inhibited by CO₂, competitively with respect to O₂. For each gas, the K_m for the reaction in which it was a substrate was similar to its K_i for the reaction it inhibited. The Arrhenius activation energy for the oxygenase reaction was 1.5 times that of the carboxylase. These characteristics are consistent with ribulose diphosphate oxygenase being the enzymatic reaction responsible for synthesizing the substrate for photorespiration and with the concept that the balance between photosynthesis and photorespiration of leaves is a reflection of the ratio between the two activities of this bi-functional enzyme.     

High specific activity ribulose 1,5-bisphosphate carboxylase-oxygenase from Nicotiana tabacum

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.     

Measurement of ribulose 1,5-bisphosphate from spinach chloroplasts

A technique has been developed for the rapid and simple measurement of ribulose 1,5-bisphosphate from isolated spinach chloroplasts. The endogenous ribulose bisphosphate was detected enzymically using (14)CO(2) and ribulose bisphosphate carboxylase/oxygenase released from the chloroplasts. Ribulose 5-phosphate kinase was inhibited with 0.4 to 0.6 millimolar 2,6-dichlorophenol-indophenol and 4 micromolar carbonyl cyanide m-chlorophenylhydrazone. Phosphoenolpyruvate carboxylase activity was low with washed chloroplasts and its labeled product, [(14)C]oxalacetate, was destroyed by heating with 1.0 n HCl at 90 C. The assay method was linear from 0.05 to 0.87 nanomoles ribulose bisphosphate per milliliter. The latter value was determined with chloroplast material having 44 micrograms of chlorophyll per milliliter. This technique was simple and direct, used less chloroplast material, yet provided results comparable to a previously described enzymic technique in which ribulose bisphosphate was determined after the precipitation of chloroplast proteins by perchloric acid.     

Models describing the kinetics of ribulose biphosphate carboxylase-oxygenase.

Equations are developed to describe the reactions of ribulose 1,5-biphosphate carboxylase—oxygenase with ribulose biphosphate (RuP₂), carbon dioxide, and oxygen. It is predicted that at the high concentrations of enzyme sites found in vivo there will be a large proportion of the total RuP₂ bound to the enzyme. The kinetic characteristics of the in vivo reactions with RuP₂ are predicted to be analogous to those which would occur in the presence of a tight-binding substrate. Equations are developed which are applicable when the enzyme is only partially activated by CO₂ and Mg²⁺. The response of carboxylase velocity to CO₂ concentration is sigmoidal when Mg²⁺ concentration is low.     

Isolation of ribulose bisphosphate carboxylase-oxygenase from non-hardened and hardened needles of Pinus sylvestris

Ribulose bisphosphate carboxylase-oxygenase, RuBP carboxylase (EC 4.1.1.39), was purified from non-hardened and hardened needles of Pinus sylvestris L. Needles were collected from pine seedlings cultivated in nutrient solution in a climate chamber from seedlings grown outdoors, and from a tree in a natural stand. The enzyme was isolated from crude extracts through quantitative precipitation in polyethylene glycol 4000 and MgCl₂, followed by sucrose gradient centrifugation in a fixed angle rotor. The purified enzyme seemed homogeneous by the criterion of (sodium dodecylsulphate) polyacrylamide gel electrophoresis. Contamination by nucleic acids was negligible. The RuBP carboxylase protein content of the gradient fractions was estimated as A₂₈₀^{1 cm}× 0.61 mg ml⁻¹. Carboxylase activities were determined in a radioactive assay at 25°C. The specific activity of RuBP carboxylase isolated from non-hardened needles was approximately 1 μmol CO₂ (mg protein)⁻¹ min⁻¹. For enzyme isolated from hardened needles collected during winter the specific activity was somewhat lower due to loss of enzyme activity during the preparation. The described two-step procedure provides a means for quantitation of the RuBP carboxylase protein in pine needles during all seasons.     

The function of the small subunits of ribulose bisphosphate carboxylase-oxygenase

When ribulose bisphosphate carboxylase-oxygenase from Synechococcus (strain RRIMP N1) was precipitated under mildly acidic conditions, most of its small subunits remained in solution. The precipitated enzyme readily redissolved at neutral pH and remained as an octamer of large subunits with some small subunits still attached. Optimum pH for this separation was 5.3 and disulfide-reducing reagents were not necessary. The fraction of small subunits removed by a single precipitation increased with increasing NaCl concentration. Catalytic activity of small subunit-depleted enzyme was linearly proportional to the fraction of small subunits remaining, while the carboxylase:oxygenase activity ratio and the affinity for CO2 remained constant. When isolated small subunits were added back to depleted enzyme preparations at neutral pH, reassociation occurred with return of catalytic activity. Under the usual assay conditions at pH 7.7, the binding constant of the small subunits was estimated to be about 10(-9) M. The small subunits also bound avidly to surfaces. However, loss of small subunits from the enzyme during the course of purification was minimal. The results are consistent with a simple model in which only those large subunits which have a small subunit bound to them are catalytically competent. Thus, an essential, even if indirect, role for the small subunits in catalysis is indicated.     

Along the trail from Fraction I protein to Rubisco (ribulose bisphosphate carboxylase-oxygenase)

This historical minireview deals with events leading to the eventual discovery of Rubisco (ribulose bisphosphate carboxylase-oxygenase). This abundant leaf protein is not only responsible for the net fixation of CO₂ in all plants, but also causes the loss of carbon through photorespiration. The latter is a special ‘problem’ of the so-called ‘C₃’ plants. The protein was first called ‘Fraction 1 protein’ before it was recognized to be the same as Rubisco. Instead of reinventing words, text as needed has been freely used from three earlier publications (Wildman and Kwanyuen 1978; Wildman 1992, 1998) This revised version was published online in June 2006 with corrections to the Cover Date.     

Properties and regulation of ribulose diphosphate carboxylase from Thiobacillus novellus

Ribulose-diphosphate carboxylase from Thiobacillus novellus has been purified to homogeneity as observed by polyacrylamide gel electrophoresis and U. V. light observation during sedimentation velocity analysis. The optimum pH for the enzyme with Tris-HCl buffers was about 8.2. Concentrations of this buffer in excess of 80 mM were inhibitory. The apparent K _m RuDP was about 14.8 M with a Hill value of 1.5, for HCO ₃ ^- the apparent K _m was about 11.7 mM with an n value of 1.18 and for Mg²⁺ about 0.61 mM. The enzyme was specific for this cation. Relatively high concentrations of either Hg²⁺ or pCMB were required before significant inhibition was observed. Activity declined slowly during a 4-hr incubation period in either 3.0 M or 8.0 M urea. Incubation for 12 hrs resulted in complete loss of activity which was not prevented by 10 mM Mg²⁺ and was not reversed by dialysis and subsequent addition of 10 mM cysteine. Polyacrylamide gel electrophoresis revealed a loss of the major band and the appearance of 2 new bands. SDS polyacrylamide gel electrophoresis gave an average M.W. of 73 500±2500 for the slower moving band and 12250 ±2500 for the faster moving. However, incubation in urea for up to 40 hrs revealed a decrease in the M.W. of the slower moving band to about 60000. The E _a for the enzyme was calculated to be about 18.85 kcal mole^-1, with the possibility of a break between 40 and 50°C. The Q ₁₀ was 3.07 between 20 to 30°C whereas between 30 to 40°C it was 3.31. Only phosphorylated compounds caused significant inhibition of enzyme activity. They included ADP, FDP, F6P, G6P, PEP, 6PG, 2-PGA, R1P, R5P and Ru5P.Abbreviations ATP adenosine-5-triphosphate - FDP fructose-1,6-diphosphate - F6P fructose-6-phosphate - G6P glucose-6-phosphate - GPDH glyceraldehyde-3-phosphate dehydrogenase - NADH nicotinamide adenine dinucleotide (reduced) - OAA oxalacetate - pCMB parachlormercuribenzoate - PEP phosphoenolpyruvate - 6PG 6-phosphogluconate - 2-PGA 2-phosphoglycerate - 3-PGA 3-phosphoglycerate - PGK 3-phosphoglyceric phosphokinase - R1P ribose-1-phosphate - R5P ribose-5-phosphate - RuDP ribulose-1,5-diphosphate - Ru5P ribulose-5-phosphate - SDS sodium dodecyl sulfate     

Ribulose diphosphate oxygenase. V. Presence in ribulose diphosphate carboxylase from Rhodospirillum rubrum

Ribulose-1,5-diphosphate carboxylase was purified fifteenfold from Rhodospirillum rubrum grown autotrophically under H₂ and CO₂. There was RuDP oxygenase activity associated with the carboxylase. The oxygenase had maximal activity at pH 9.4. Although these bacterial RuDP oxygenase and carboxylase activities were cold labile, activity could not be restored by treatment at 50° in the presence of Mg⁺⁺ and a sulfhydryl reagent, in contrast to results with the enzyme from eukaryotes.     

Conditions leading to precipitation of ribulose bisphosphate carboxylase-oxygenase differ from those leading to enzyme activation

The relation between conditions leading to precipitation and/or activation of Ru-P₂ carboxylase have been explored in order to test the hypothesis that conformational changes leading to precipitation might be identical to those which are presumed to lead to enzyme activation. From the results of kinetic and solubility studies, we conclude that this hypothesis is not valid, since changes in solubility of Ru-P₂ carboxylase occur ten times as fast as changes in enzyme kinetics.Abbreviations Ru-P₂ ribulose 1,5-bisphosphate - PVP polyvinylpyrrolidone - DTE dithioerythritol - Bicine N,N-bishydroxy-2-ethylglycine     

Conditions leading to precipitation of ribulose bisphosphate carboxylase-oxygenase differ from those leading to enzyme activation

The relation between conditions leading to precipitation and/or activation of Ru-P₂ carboxylase have been explored in order to test the hypothesis that conformational changes leading to precipitation might be identical to those which are presumed to lead to enzyme activation. From the results of kinetic and solubility studies, we conclude that this hypothesis is not valid, since changes in solubility of Ru-P₂ carboxylase occur ten times as fast as changes in enzyme kinetics.     

Structural and regulatory studies on the ribulose bisphosphate carboxylase-oxygenase of Anabaena cylindrica

Ribulose 1,5-bisphosphate (RuBP) carboxylase-oxygenase fromthe cyanobacterium Anabaena cylindrica has been purified tohomogeneity by the criterion of polyacrylamide gel electrophoresisand shown to consist of two types of subunits of molecular weights51K (large) and 12K (small). The enzyme is of the higher planttype and probably consists of 8 large plus small subunits. Isoelectricfocusing of the S-carboxymethylated protein in 8 M urea revealeda profile of consisting of 3 major polypeptides plus 1 minorpolypeptide. Some characteristics of the carboxylase and oxygenasereactions were studied using simultaneous measurements of bothactivities. Pyridoxal 5'-phosphate inhibited both activitiesequally. Neither the carboxylase nor oxygenase reaction wasaffected by glutamate (5 mM), although 6-phosphogluconate andfructose 1,6-bisphosphate inhibited both reactions. RuBP oxygenasewas more sensitive to 6-phosphogluconate (0.5 and 1.0 mM) thanRuBP carboxylase. Marked changes in the oxygenase to carboxylaseactivity ratio of the purified enzyme were effected by homologousantiserum (which preferentially inhibited carboxylation). ¹Present address: Institute of Applied Microbiology, Universityof Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received May 22, 1980; )     

Localization and properties of ribulose diphosphate carboxylase from castor bean endosperm

A substantial portion of the ribulose 1,5-diphosphate carboxylase activity in the endosperm of germinating castor beans (Ricinus communis var. Hale) is recovered in the proplastid fraction. The partially purified enzyme shows homology with the enzyme from spinach (Spinacia oleracea) leaves, as evidenced by its reaction against antibodies to the native spinach enzyme and to its catalytic subunit. The enzyme from the endosperm of castor beans has a molecular weight of about 500,000 and, with the exception of a higher affinity for ribulose 1,5-diphosphate, has similar kinetic properties to the spinach enzyme. The castor bean carboxylase is inhibited by oxygen and also displays ribulose 1,5-diphosphate oxygenase activity with an optimum at pH 7.5.