Distinct reactions catalyzed by bacterial and yeast trans-aconitate methyltransferases

The trans-aconitate methyltransferase from the bacterium Escherichia coli catalyzes the monomethyl esterification of trans-aconitate and related compounds. Using two-dimensional (1)H/(13)C nuclear magnetic resonance spectroscopy, we show that the methylation is specific to one of the three carboxyl groups and further demonstrate that the product is the 6-methyl ester of trans-aconitate (E-3-carboxy-2-pentenedioate 6-methyl ester). A similar enzymatic activity is present in the yeast Saccharomyces cerevisiae. Although we find that yeast trans-aconitate methyltransferase also catalyzes the monomethyl esterification of trans-aconitate, we identify that the methylation product of yeast is the 5-methyl ester (E-3-carboxyl-2-pentenedioate 5-methyl ester). The difference in the reaction catalyzed by the two enzymes may explain why a close homologue of the E. coli methyltransferase gene is not found in the yeast genome and furthermore suggests that these two enzymes may play distinct roles. However, we demonstrate here that the conversion of trans-aconitate to each of these products can mitigate its inhibitory effect on aconitase, a key enzyme of the citric acid cycle, suggesting that these methyltransferases may achieve the same physiological function with distinct chemistries.     

Inhibition and inactivation of liver aldolase

Substrate analogs xylulose 1,5-bisphosphate, glucitol 1,6-bisphosphate, α-2,5-anhydroglucitol 1,6-bisphosphate, α-, β-methyl fructofuranoside 1,6-bisphosphate, ribulose 1,5-bisphosphate, ribulose 5-phosphate, and ribose 5-phosphate and inactivating agents 1-chloro-2, 4-dinitrobenzene, 4-hydroxymercuribenzoate, and pyridoxal phosphate were examined for their effects on liver aldolase. These studies support the use of the β-anomer and acyclic form as substrate. They also suggest that the liver enzyme active site is similar to the muscle enzyme but with a much weaker 6-phosphate binding site.     

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.     

Regulation of photosynthesis in nitrogen deficient wheat seedlings

Nitrogen effects on the regulation of photosynthesis in wheat (Triticum aestivum L., cv Remia) seedlings were examined. Ribulose 1,5-bisphosphate carboxylase/oxygenase was rapidly extracted and tested for initial activity and for activity after incubation in presence of CO₂ and Mg²⁺. Freeze clamped leaf segments were extracted for determinations of foliar steady state levels of ribulose 1,5-bisphosphate, triose phosphate, 3-phosphoglycerate, ATP, and ADP. Nitrogen deficient leaves showed increased ATP/ADP and triose phosphate/3-phosphoglycerate ratios suggesting increased assimilatory power. Ribulose 1,5-bisphosphate levels were decreased due to reduced pentose phosphate reductive cycle activity. Nevertheless, photosynthesis appeared to be limited by ribulose 1,5-bisphosphate carboxylase/oxygenase, independent of nitrogen nutrition. Its degree of activation was increased in nitrogen deficient plants and provided for maximum photosynthesis at decreased enzyme protein levels. It is suggested that ribulose 1,5-bisphosphate carboxylase/oxygenase activity is regulated according to the amount of assimilatory power.     

不同发育时期追施氮肥对冬小麦旗叶中光合酶活性的影响

小麦开花后,随着旗叶的衰老,旗叶中1,5-二磷酸核酮糖羧化酶（RuBPC）、磷酸烯醇式丙酮酸羧化酶（PEPC）和乙醇酸氧化酶（GO）活性呈下降趋势。随着追施氮肥时期的推迟,光合酶活性呈增加趋势,这意味着氮肥追施时间后移有利于提高小麦光合速率。在旗叶衰老后期,大穗型品种小麦旗叶中光合酶活性略高于多穗型品种小麦。     

Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis

The epsilon-amino group of Lys-166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase was postulated as the essential base which initiates catalysis by abstracting the proton at C-3 of ribulose 1,5-bisphosphate (Hartman, F. C., Soper, T. S., Niyogi, S. K., Mural, R. J., Foote, R. S., Mitra, S., Lee, E. H., Machanoff, R., and Larimer, F. W. (1987) J. Biol. Chem. 262, 3496-3501). To scrutinize this possibility, the site-directed Gly-166 mutant, totally devoid of ribulosebisphosphate carboxylase activity, was examined for its ability to catalyze each of three partial reactions. When carbamylated at Lys-191 (i.e. activated with CO2 and Mg2+), wild-type enzyme catalyzed the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon reaction intermediate of the carboxylase reaction (Pierce, J., Andrews, T. J., and Lorimer, G. H. (1986a) J. Biol. Chem. 261, 10248-10256). Likewise, when carbamylated at Lys-191, the Gly-166 mutant also catalyzed the hydrolysis of this reaction intermediate. The carbamylated wild type catalyzed the enolization of ribulose 1,5-bisphosphate as indicated by the transfer of 3H radioactivity from [3-3H]ribulose, 1,5-bisphosphate to the medium. However, even when carbamylated at Lys-191, the mutant protein did not catalyze the enolization of ribulose 1,5-bisphosphate. Additionally, unlike the decarbamylated wild-type enzyme, which catalyzed the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate in the absence of Mg2+, the mutant protein was inactive in this partial reaction. These properties exclude the epsilon-amino group of Lys-166 as an obligatory participant in the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate. In contrast, these properties are consistent with the epsilon-amino group of Lys-166 functioning as an acid-base catalyst in the enolization of ribulose 1,5-bisphosphate (when the enzyme is carbamylated) and in the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate (when the enzyme is decarbamylated). Alternatively, Lys-166 may stabilize the transition states of these two partial reactions.     

Measurement and preservation of the in vivo activation of ribulose 1,5-bisphosphate carboxylase in leaf extracts

Photosynthetic carbon fixation is regulated in the chloroplast by the amount of ribulose 1,5-bisphosphate carboxylase which is activated. The activated carboxylase was preserved in detached leaves (barley, maize, soybean, spinach, wheat) for 90 min when stored on ice. With leaf extracts stored at 2°C, the amount of activated enzyme, representing that originally in the leaf, as well as the fully activated enzyme, formed by incubation of leaf extracts with Mg²⁺ and bicarbonate, both slowly declined in activity. However, for each activity this decline was proportional such that the ratio (percent activation) appeared constant. No change was observed in activation of the enzyme during the brief time of leaf homogenization. Optimal conditions (Mg²⁺, incubation time) for measurement of leaf activation of ribulose bisphosphate carboxylase vary depending on the plant.     

Effects of growth temperature on photosynthetic carbon metabolism in green plants II. Photsoynthetic 14CO2-incorporation in plants acclimatized to varied temperatures

During photosynthetic ¹⁴CO₂-fixation, leaves of plants suchas wheat, the broad bean and spinach, which had been acclimatizedto high temperature (20–25?C), incorporated much moreradioactivity into sucrose, and less into glycine and serinein comparison with similar plants grown in the cold (mean temperature,5–7?C). Radioactivities incorporated into glycine and serine greatlydescreased on the addition of -hydroxyethylsulfonate or on theremoval of oxygen from the atmosphere, indicating that thesecompounds are synthesized through the glycolate pathway. In leaves of wheat grown under low temperatures, relativelyhigh radioactivity was detected in ribulose 1,5-diphosphateamong the photosynthetic ¹⁴CO₂-fixation products, whereas practicallyno radioactivity was detected in this compound in leaves ofwheat which had been acclimatized to high temperatures. We assumedthat the carboxylation reaction of ribulose 1,5-diphosphateis suppressed in plants acclimatized to low temperatures. It was further inferred that the C-2 and C-2 moiety of ribulose1,5-diphosphate accumulating as a result of suppression of carboxylationis converted to glycine and serine through the glycolate pathway. The possibility was also discussed that during photosyntheticCO₂-fixation in wheat leaves at least a part of the C₆-compoundformed by the carboxylation of ribulose 1,5-diphosphate is directlyconverted to sugar phosphate. ¹Part of this investigation was reported at the 2nd InternationalCongress on Photosynthesis Research at Stresa, Italy, June 1971.This paper is based on a dissertation submitted by S.S. to theFaculty of Science, the University of Tokyo, in partial fulfilmentof the requirements for a Ph.D. degree. ²Present address: Department of Botany, Faculty of Science,University of Tokyo, Tokyo, Japan. (Received July 20, 1973; )     

Ribulose 1,5-bisphosphate carboxylase from the thermophilic, acidophilic alga, Cyanidium caldarium (Geitler). Purification, characterisation and thermostability of the enzyme.

An an initial stage in the study of proteins from thermophilic algae, the enzyme ribulose 1,5-bisphosphate carboxylase 2-phospho-D-glycerate carboxylyase (dimerizing, EC 4.1.1.39) was purified 11-fold from the thermophilic alga Cyandium caldarium, with a 24% recovery. This purified enzyme appeared homogeneous on polyacrylamide gels and could be dissociated into two subunit types of molecular weights 55,000 and 14,900. The optimal assay temperature was 42.5 degrees C, whilst enzyme purified from Chlorella spp. showed maximum activity at 35 degrees C. The thermostability of Cyanidium ribulose 1,5-bisphosphate carboxylase was considerably greater than that of the Chlorella enzyme, and the presence of Mg2+ and HCO-3 further enhanced this heat stability. A break in the Arrhenius plot occured at 20 degrees C for Chlorella ribulose 1,5-bisphosphate carboxylase and 36 degrees C for the enzyme from Cyanidium. It is suggested that the thermostability of Cyanidium ribulose 1,5-bisphosphate carboxylase is a result of an inherent stability of the enzyme molecule which permits efficient CO2 fixation at high temperatures but results in low activity in the mesophilic temperature range.     

Daphnetin, one of coumarin derivatives, is a protein kinase inhibitor.

Protein kinases play key roles in the control of cell proliferation, differentiation and metabolism. In this work, we studied the effect of coumarin and its derivatives, including daphnetin, esculin, 2-OH-coumarin, 4-OH-coumarin and 7-OH-coumarin, on the activity of protein kinases. It was found that, in these compounds, only daphnetin was a protein kinase inhibitor. This compound inhibited tyrosine-specific protein kinase, EGF receptor (IC(50) = 7.67 microM), and serine/threonine-specific protein kinases, including cAMP-dependent protein kinase (PKA) (IC(50) = 9.33 microM) and protein kinase C (PKC) (IC(50) = 25.01 microM) in vitro. The inhibition of EGF receptor tyrosine kinase by daphnetin was competitive to ATP and non-competitive to the peptide substrate. The inhibition of EGF-induced tyrosine phosphorylation of EGF receptor by daphnetin was not observed in human hepatocellular carcinoma HepG2 cells. The structural comparison of daphnetin with coumarin and other coumarin derivatives suggests that the hydroxylation at C8 may be required for daphnetin acting as a protein kinase inhibitor.     

The Activation State of Ribulose 1,5-bisphosphate Carboxylase in Maize Leaves in Dark and Light

A spectrophotometric procedure for assay of initial and totalactivity of ribulose 1,5-bisphosphate carboxylase in maize leaveswas established. The extraction of the crude enzyme from maizeleaf tissue, which was prefrozen in liquid nitrogen, desaltingof the extract, and assay of the enzyme was completed within3 min. From experiments adding deactivated ribulose 1,5-bisphosphatecarboxylase to the leaf tissue prior to extraction it was estimatedthat the maximum extent of activation during extraction, desaltingand assay was 8%. In predarkened leaves the enzyme showed 67to 84% of maximal activation while in preilluminated leavesthe enzyme showed 89 to 98% of maximal activation. These resultsindicate that deactivation of the enzyme in the dark is nota reason for the previous finding of a transient peak of ribulose1,5-bisphosphate in maize leaves during induction of photosynthesis[Usuda (1985) Plant Physiol. 78: 859–864]. This transientincrease in the substrate level upon illumination might be explainedby the presence of an unknown negative effector for ribulose1,5-bisphosphate carboxylase in vivo in leaf tissue in the dark,or limiting CO₂ supply to the enzyme during the induction period. (Received May 30, 1985; Accepted August 16, 1985)     

Dynamic, ligand-dependent conformational change triggers reaction of ribose-1,5-bisphosphate isomerase from Thermococcus kodakarensis KOD1

Ribose-1,5-bisphosphate isomerase (R15Pi) is a novel enzyme recently identified as a member of an AMP metabolic pathway in archaea. The enzyme converts d-ribose 1,5-bisphosphate into ribulose 1,5-bisphosphate, providing the substrate for archaeal ribulose-1,5-bisphosphate carboxylase/oxygenases. We here report the crystal structures of R15Pi from Thermococcus kodakarensis KOD1 (Tk-R15Pi) with and without its substrate or product. Tk-R15Pi is a hexameric enzyme formed by the trimerization of dimer units. Biochemical analyses show that Tk-R15Pi only accepts the α-anomer of d-ribose 1,5-bisphosphate and that Cys(133) and Asp(202) residues are essential for ribulose 1,5-bisphosphate production. Comparison of the determined structures reveals that the unliganded and product-binding structures are in an open form, whereas the substrate-binding structure adopts a closed form, indicating domain movement upon substrate binding. The conformational change to the closed form optimizes active site configuration and also isolates the active site from the solvent, which may allow deprotonation of Cys(133) and protonation of Asp(202) to occur. The structural features of the substrate-binding form and biochemical evidence lead us to propose that the isomerase reaction proceeds via a cis-phosphoenolate intermediate.     

Interactions between ribulose-1,5-bisphosphate carboxylase and stromal metabolites. II. Corroboration of the role of this enzyme as a metabolite buffer

The capacity of ribulose-1,5-bisphosphate carboxylase to bind reversibly chloroplast metabolites which are the substrates for both thylakoid and stromal enzymes was assessed using spinach chloroplasts and chloroplast extracts and with pure wheat ribulose-1,5-bisphosphate carboxylase. Measurements of the rate of coupled electron flow to methyl viologen in ‘leaky’ chloroplasts (which retained the chloroplast envelope and stromal enzymes but which were permeable to metabolites) and also with broken chloroplasts and washed thylakoids were used to study the effects of binding ADP and inorganic phopshate to ribulose-1,5-bisphosphate carboxylase. The presence of ribulose-1,5-bisphosphate carboxylase significantly altered the values obtained for apparent K_m for inorganic phosphate and ADP of coupled electron transport. The K_m (P_i) in washed thylakoids was 60–80 μM, in ‘leaky’ chloroplasts it was increased to 180–200 μM, while in ‘leaky’ chloroplasts preincubated with KCN and ribulose 1,5-bisphosphate the value was decreased to 40–50 μM. Similarly, the K_m (ADP) of coupled electron transport in washed thylakoids was 60–70 μM, in ‘leaky’ chloroplasts it was 130–150 μM and with ‘leaky’ chloroplasts incubated in the presence of KCN and ribulose 1,5-bisphosphate a value of 45–50 μM was obtained. The ability of ribulose 1,5-bisphosphate carboxylase to reduce the levels of free glycerate 3-phosphate in the absence of ribulose 1,5-bisphosphate was examined using a chloroplast extract system by varying the concentrations of stromal protein or purified ribulose 1,5-bisphosphate carboxylase. The effect of binding glycerate 3-phosphate to ribulose-1,5-bisphosphate carboxylase on glycerate 3-phosphate reduction was to reduce both the rate an the amount of NADPH oxidation for a given amount of glycerate 3-phosphate added. The addition of ribulose 1,5-bisphosphate reinitiated NADPH oxidation but ATP or NADPH did not. Incubation of purified ribulose-1,5-bisphosphate carboxylase with carboxyarabinitolbisphosphate completely inhibited the catalytic activity of the enzyme and decreased inhibition of glycerate-3-phosphate reduction. Two binding sites with different affinities for glycerate 3-phosphate were observed with pure ribulose-1,5-bisphosphate carboxylase.     

Pyrogalloloestrogens: enzymic methylation of pyrogalloloestrogens and interaction of pyrogalloloestrogens with the enzymic methylation of catecholamines in rat liver in vitro.

During incubation of 2,4-dihydroxyoestrone with the 105000 X g supernatant of rat liver in the presence of S-adenosyl-[Me-14C]methionine, the formation of radioactive mono- as well as dimethyl ether derivatives was demonstrated. The products were identified as: 2,4-dihydroxyoestrone 2-methyl ether, 2,4-dihydroxyoestrone 3-methyl ether, 2,4-dihydroxyoestrone 4-methyl ether, 2,4-dihydroxyoestrone 2,3-dimethyl ether, 2,4-dihydroxyoestrone 2,4-dimethyl ether and 2,4-dihydroxyoestrone 3,4-dimethyl ether. The monomethyl ethers were the main products; within this group the 3-methyl ether of 2,4-dihydroxyoestrone was the main metabolite. Among the dimethyl ether derivatives, the 2,4-dihydroxyoestrone 2,3-dimethyl ether represented the quantitatively most important product. When 2,4-dihydroxyoestrone 2-methyl ether was incubated under the same conditions, 2,4-dihydroxyoestrone 2,3- as well as 2,4-dimethyl ether was formed. The 2,3-dimethyl ether was again the main metabolite. The incubation of 2,4-dihydroxyoestrone 4-methyl ether yielded the 2,4- and 3,4-dimethyl ethers, the first being the main product. In contrast, the 3-methyl ether of 2,4-dihydroxyoestrone was not further methylated by the catechol methyltransferase preparation. In further experiments, the effect of the pyrogalloloestrogen and its monomethyl ether derivatives on the enzymatic methylation of catecholamines was investigated. It was demonstrated that the methylation of adrenalin and dopamine was competitively inhibited by 2,4-dihydroxyoestrone and the 2,4-dihydroxyoestrone monomethyl ethers. Only a weak inhibitory effect was observed with the 3- and 4-monomenthyl ethers (Ki values 200 and 160muM). The unsubstituted pyrogalloloestrogen produced a marked inhibition (Ki value 50muM), but the strongest inhibition was found with the 2-monomethyl ether of 2,4-dihydroxyoestrone (Ki value 14muM). The extent of inhibition caused by the addition of the 2-monomethyl ether of 2,4-dihydroxyoestrone was thereby in the same range as the inhibition caused by pyrogallol and the catecholoestrogens.     

Ribulose 1,5-bisphosphate carboxylase-oxygenase. I. Structural, immunochemical and catalytic properties

Some structural, immunochemical and catalytic properties are examined for ribulose 1,5-bisphosphate carboxylase-oxygenase from various cellular organisms including bacteria, cyanobacteria, algae and higher plants. The native enzyme molecular masses and the subunit polypeptide compositions vary according to enzyme sources. The molecular masses of the large and small subunits from different cellular organisms, on the other hand, show a relatively high homology due to their well-conserved primary amino acid sequence, especially that of the large subunit. In higher plants, the native enzyme and the large subunit are recognized by the antibodies raised against either the native or large subunit, whereas the small subunit apparently cross-reacts only with the antibodies directed against itself. A wide diversity exists, however, in the serological response of the native enzyme and its subunits with antibodies directed against the native enzyme or its subunits from different cellular organisms. According to numerous kinetic studies, the carboxylase and oxygenase reactions of the enzyme with ribulose 1,5-bisphosphate and carbon dioxide or oxygen require activation by carbon dioxide and magnesium prior to catalysis with ribulose 1,5-bisphosphate and carbon dioxide or oxygen. The activation and catalysis are also under the regulation of other metal ions and a number of chloroplastic metabolites. Recent double-labeling experiments using radioactive ribulose 1,5-bisphosphate and 14CO2 have elucidated the carboxylase/oxygenase ratios of the enzymes from different organisms. Another approach, i.e., genetic experiments, has also been used to examine the modification of the carboxylase/oxygenase ratio.     

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios.

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) FROM SPINACH CHLOROPLASTS IS STRONGLY REGULATED BY THE RATIO OF NADPH/NADP+, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP+ ratio. With a ratio of NADPH/NADP+ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited. This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP+ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions. Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP+ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP+. It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP+ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolic unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP+ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.     

Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.