Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum

Abstract Phosphoenolpyruvate (PEP) carboxykinase is present in crude extracts of Corynebacterium glutamicum grown on both glucose and lactate. Preparation of PEP carboxykinase free from interfering PEP carboxylase and oxaloacetate decarboxylase showed an absolute dependence on divalent manganese and IDP for activity in the oxaloacetate (OAA) formation. Other diphosphate nucleotides could not substitute for IDP. The enzyme activity displayed Michaelis-Menten kinetics for the substrates PEP, IDP, KHCO₃, OAA and ITP with a K _m of 0.7 mM, 0.4 mM, 12 mM, 1.0 mM, and 0.5 mM, respectively. At the optimum pH of 6.6, 850 nmol of OAA were formed per min per mg of protein. ATP inhibited PEP carboxykinase in the OAA forming reaction for 60% at 0.1 mM, indicating that the enzyme mainly functions in gluconeogenesis.     

Bicarbonate transport and extracellular carbonic anhydrase in marine diatoms

In this article, we present new laboratory results examining the relative importance of HCO⁻₃ transport and extracellular carbonic anhydrase (eCA) in 17 marine diatom species. We observed significant variability in both HCO⁻₃ transport and eCA expression across a range of diatom species with different cell morphologies. All species we examined took up HCO⁻₃ through a direct transport mechanism, with the fraction of HCO⁻₃ transport ranging from 40 to 95% of total C uptake. eCA expression also varied significantly, with catalytic enhancement factors ranging approximately 10-fold among species. There was a significant positive correlation between HCO⁻₃ transport and eCA expression among the test species. However, neither HCO⁻₃ transport nor eCA expression was significantly correlated to cell growth rates or surface area to volume ratios. We did observe weak positive trends between the ratio of C demand:supply and HCO⁻₃ utilization/eCA expression, but these were not statistically significant. We are thus unable to provide a mechanistic explanation for the apparent variability in HCO⁻₃ transport and eCA expression in marine diatoms. This variability may, nonetheless, have important implications for the physiological ecology of oceanic diatoms.     

On the participation of chloride in bicarbonate-induced reversal of anion inhibition of photosystem II electron transport in spinach thylakoids

Inhibition of electron transport through photosystem II (PS II) by formate (HCO⁻₂) or nitrite (NO⁻₂) in the presence or absence of chloride ions was studied. The inhibition induced by HCO⁻₂ or NO⁻₂ is overcome by HCO⁻₃ more in the presence, than in the absence of Cl⁻. The data on electron transport are supported by chlorophyll a fluorescence measurements. In experiments. In experiments in which water oxidation was blocked. Cl⁻ was found to facilitate electron transport between bound quinone A (Q⁻_A) and the plastoquinone (PQ) pool. It can thus be concluded that in addition to the well known site of action of Cl⁻ on water oxidation, another site of Cl⁻ action is between Q⁻_A and the PQ pool.     

Effects on acid-base balance, methaemoglobinemia and nitrogen excretion of European eel after exposure to elevated ambient nitrite

Haemoglobin, methaemoglobin, blood nitrite concentration and acid-base balance were measured in European eel Anguilla anguilla following exposure to 0 (control), 0·142, 0·356, 0·751 and l·549 mM nitrite in fresh water for 24 h. Blood GOT (glutamate oxaloacetate transaminase) and GPT (glutamate pyruvate transaminase) activities and whole animal ammonia-N and urea-N excretions were also measured. Blood nitrite, methaemoglobin, PO ₂ (oxygen partial pressure), GOT, and whole animal ammonia-N excretion and urea-N excretion increased directly with increasing ambient nitrite concentrations, whereas blood pH, PCO ₂, and HCO⁻₃ were inversely related to ambient nitrite concentration. An accumulation of nitrite in the blood of A. anguilla following 24 h exposure to elevated ambient nitrite as low as 0·751 mM increased its blood methaemoglobin, PO ₂, GOT and nitrogen excretion, but decreased its PCO ₂ (carbon dioxide partial pressure), HCO⁻₃ and functional haemoglobin.     

BICARBONATE UTILIZATION BY MARINE PHYTOPLANKTON SPECIES

The contribution of bicarbonate to total dissolved inorganic carbon (DIC) utilization was investigated using 18 marine phytoplankton species, including members of Bacillariophyceae, Dinophyceae, Prymnesiophyceae, and Raphidophyceae, under carbon-replete or -limited conditions. Extracellular carbonic anhydrase (CA) was assayed as an indicator of extracellular CA-catalyzed HCO⁻₃ utilization. For some species, extracellular CA was constitutive, in others activity was detected under conditions of carbon limitation, and in others, even under carbon-limited conditions, activity was not detected. In species without extracellular CA, direct HCO⁻₃ uptake was investigated using a pH drift technique in a closed system, DIC measurements, and the use of the anion exchange inhibitor 4'4'-diisothiocyanatostilbene-2,2-disulfonic acid (DLDS). Three of these species (Chaetoceros compressus, Thalassiosira pseudonana, and Glenodinium foliaceum) gave a pH drift not inhibited by DIDS, but cultures of Chrysochromulina kappa, Gephrocapsa oceanica, and Coccolithus pelagicus, in which DLDS inhibited DIC uptake, did not give a pH drift. This result shows that direct HCO₃⁻ transport may occur by an anion exchange-type mechanism in some species but not others. Of the eighteen species investigated, only Heterosigma akashiwo did not have the potential for direct uptake or extracellular CA-catalyzed HCO⁻₃ utilization.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: pathways of C4 acid decarboxylation in bundle sheath cells of Urochloa panicoides

The mechanism of C4 acid decarboxylation was studied in bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C4 plant. Added malate was decarboxylated to give pyruvate and this activity was often increased by adding ADP. Added oxaloacetate or aspartate plus 2-oxoglutarate (which produce oxaloacetate via aspartate aminotransferase) gave little metabolic decarboxylation alone but with added ATP there was a rapid production of PEP. For this activity ADP could replace ATP but only when added in combination with malate. In addition, the inclusion of aspartate plus 2-oxoglutarate with malate plus ADP often increased the rate of pyruvate production from malate by more than twofold. Experiments with respiratory chain inhibitors showed that the malate-dependent stimulation of oxaloacetate decarboxylation (PEP production) was probably due to ATP generated during the oxidation of malate in mitochondria. We could provide no evidence that photophosphorylation could serve as an alternative source of ATP for the PEP carboxykinase reaction. We concluded that both PEP carboxykinase and mitochondrial NAD-malic enzyme contribute to C4 acid decarboxylation in these cells, with the required ATP being derived from oxidation-linked phosphorylation in mitochondria.     

Lysine 213 and histidine 233 participate in Mn(II) binding and catalysis in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO2 and plays a key role in gluconeogenesis. This enzyme also has oxaloacetate decarboxylase and pyruvate kinase-like activities. Mutations of PEP carboxykinase have been constructed where the residues Lys213 and His233, two residues of the putative Mn2+ binding site of the enzyme, were altered. Replacement of these residues by Arg and by Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn2+ binding affinity. Lower PEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against urea denaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in V(max) for the primary reaction relative to that for the wild-type enzyme. V(max) values for the oxaloacetate decarboxylase and pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in the mutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn2+ and PEP. The results are consistent with Lys213 and His233 being at the Mn2+ binding site of S. cerevisiae PEP carboxykinase and the Mn2+ affecting the PEP interaction. The different effects of mutations in V(max) for the main reaction and the secondary activities suggest different rate-limiting steps for these reactions.     

Regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase activity in adult Fasciola hepatica (Trematoda).

F. hepatica pyruvate kinase and phosphoenolpyruvate (PEP) carboxykinase were found to have properties of regulatory enzymes in the dissimilation of PEP and the control of metabolic flow. Mn²⁺ and K⁺ were required for pyruvate kinase activity. In the presence of fructose-1, 6-diphosphate (FDP), Mg²⁺ could substitute for Mn²⁺. FDP caused a 4-fold increase in the Mn²⁺ activated pyruvate kinase activity. This was accompanied by a 12-fold decrease in apparent K_m(PEP) and a 3-fold decrease in apparent K_{m (ADP)}. ATP markedly inhibited F. hepatica pyruvate kinase, but this inhibition was relieved by FDP. Estimates of metabolic levels indicated that the pyruvate kinase is saturated with PEP and ADP in vivo, but will be highly sensitive to fluctuations in the physiological concentrations of FDP and ATP. NADH doubled the activity of the PEP carboxykinase reaction and decreased the apparent K_{m (PEP)} for this enzyme 3-fold. While the maximal activity of the PEP carboxykinase reaction was substantially higher than the pyruvate kinase reaction, the steady state concentration of PEP suggests that the PEP carboxykinase will not be saturated with this substrate.     

Calcification in aquatic plants

Abstract. The CaCO₃ deposits of aquatic plants may be intra-, inter- and extracellular. Calcification is mainly the result of photosynthetic CO₂ or HCO⁻₃ assimilation. This raises the local pH and CO²⁻₃ concentration resulting from shifts in the dissolved inorganic carbon equilibrium, due to either net CO₂ depletion as in Halimeda or localized OH⁻ efflux (or H⁺ influx) as in Chara. The plant cell wall may be important in CaCO₃ nucleation by acting as an epitaxial substratum or template, or by creating a microenvironment enriched in Ca²⁺ compared to Mg²⁺. Hypotheses on the reason for the lack of calcification in many aquatic plants are presented.     

Effects of phosphoenol pyruvate carboxylase deficiency on metabolism and lysine production in Corynebacterium glutamicum

The phosphoenol pyruvate carboxylase gene (ppc) of lysine-producing Corynebacterium glutamicum and C. lactofermentum strains was inactivated by marker exchange mutagenesis. The mutants lacked completely phosphoenol pyruvate carboxylase (PEP carboxylase) activity, but grew in minimal medium containing glucose as the sole carbon source. In addition, the ppc ^– strains produced equivalent titers of lysine in shake flasks and in 10-l fermentation experiments as their parent strains. To address the question of how ppc ^– Corynebacterium strains generate oxaloacetate (OAA) for their own metabolism as well as for high-level lysine production, we measured the activities of enzymes leading to OAA synthesis. Whereas pyruvate carboxylase activity was not detected in any of the strains, phosphoenol pyruvate carboxykinase (PEP carboxykinase) activity was found to be significantly higher in C. glutamicum ppc mutants compared to the parent strains. On the other hand, PEP carboxykinase activity in C. lactofermentum was essentially absent. As glyxylate cycle enzymes are strongly repressed by glucose, they are not likely to compensate for the lack of PEP carboxylase activity. PEP carboxykinase, among several candidates, could play this role. Correspondence to: M. Gubler     

Interfering mechanism of sodium bicarbonate on spore germination of Bacillus stearothermophilus

Spore germination of Bacillus stearothermophilus was progressively inhibited as the concentrations of sodium bicarbonate (NaHCO₃) in the germination media increased from 0% to 1·0% (w/v). The inhibitory effect of NaHCO₃ was attributed to the release of HCO⁻₃ and its alkaline properties, each of which played a different role. At low concentrations (< 0·3%), the inhibitory effect of NaHCO₃ was mainly due to bicarbonate. As NaHCO₃ increased from 0·3% to higher concentrations, the effect of HCO⁻₃ reached a plateau while the alkalinating effect became the more dominant inhibitory factor. Fourier transform infrared (FTIR) analysis reveals that sodium bicarbonate reacted with the carboxyl group (1570 cm⁻¹) of some acidic amino-acid residues of protein in the spore, leading to a less orientated structure. A shift of two units towards the longer frequency for carboxyl groups indicates that a stronger interaction was formed between the carboxyl group and the Na⁺ ion. The largest ratio of peak height between the absorbance of carboxylate (1570 cm⁻¹) and of amide II (1546 cm⁻¹) of spores after pretreatment with 0·3% sodium bicarbonate reflects the biggest structural alterations of keratin-like proteins in the spore. The role of NaHCO₃ in enhancing the sporicidal effect of glutaraldehyde is discussed.     

Nitrate and nitrite reduction by alfalfa root nodules: Accumulation of nitrite in Rhizobium melioti bacteroids and senescence of nodules

Addition of NO⁻₃ rapidly induced senescence of root nodules in alfalfa ( Medicago sativa L. cv. Aragon). Loss of nodule dry matter began at the lowest NO⁻₃ concentration (10 m M ) but degradation of bacteroid proteins was only detected when nodules were supplied with NO⁻₃ concentrations above 20 m M .
Bacteroids from Rhizobium meliloti contained high specific activities of nitrate reductase (NR) and nitrite reductase (NiR). Both enzymes were presumably substrate-induced although substantial enzyme activities were present in the absence of NO⁻₃ Typical specific activities for soluble NR and NiR of bacteroids under NO⁻₃ free conditions were 1.2 and 1.4 μmol (mg protein)⁻¹h⁻¹, respectively. In the presence of NO⁻₃, the specific activity of NR was considerably greater than that of NiR, thus causing NO⁻₂ accumulation in bacteroids. Nitrite levels in the bacteroids were linearly correlated with specific activities of NR and NiR, indicating that NO⁻₂ is formed by bacteroid NR and that this NO⁻₂ in turn, induces bacteroid NiR. Accumulation of NO⁻₂ within bacteroids also indicates that NO⁻₂ inhibits nodule activity after feeding plants with NO⁻₃     

Stimulation of respiration by Pb2+ in detached leaves and mitochondria of C3 and C4 plants

The exposure of detached leaves of C₃ plants (pea, barley) and C₄ plant (maize) to 5 m M Pb (NO₃)₂ for 24 h caused a reduction of their photosynthetic activity by 40–60%, whereas the respiratory rate was stimulated by 20–50%. Mitochondria isolated from Pb²⁺-treated pea leaves oxidized substrates (glycine, succinate, malate) at higher rates than mitochondria from control leaves. The respiratory control (RCR) and the ADP/O ratio were not affected. Pb²⁺ caused an increase in ATP content and the ATP/ADP ratio in pea and maize leaves. Rapid fractionation of barley protoplasts incubated at low and high CO₂ conditions, indicated that the increased ATP/ADP ratio in Pb²⁺-treated leaves resulted mainly from the production of mitochondrial ATP. The measurements of membrane potential of mitochondria with a TPP⁺-sensitive electrode further showed that mitochondria isolated from Pb²⁺-treated leaves had at least as high membrane potential as mitochondria from control leaves. The activity of NAD-malate dehydrogenase in the protoplasts from barley leaves treated with Pb²⁺ was 3-fold higher than in protoplasts from control leaves. The activities of photorespiratory enzymes NADH-hydroxypyruvate reductase and glycolate oxidase as well as of NAD-malic enzyme were not affected. The presented data indicate that stimulation of respiration in leaves treated by lead is in a close relationship with activation of malate dehydrogenase and stimulation of the mitochondrial ATP production. Thus, respiration might fulfil a protective role during heavy metal exposure.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: photosynthetic activities of isolated bundle sheath cells from Urochloa panicoides

A method has been developed for rapidly preparing bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase-type C4 plant. These cells catalyzed both HCO3(-)- and oxaloacetate-dependent oxygen evolution; oxaloacetate-dependent oxygen evolution was stimulated by ATP. For this activity oxaloacetate could be replaced by aspartate plus 2-oxoglutarate. Both oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution were accompanied by PEP production and both were inhibited by 3-mercaptopicolinic acid, an inhibitor of PEP carboxykinase. The ATP requirement for oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution could be replaced by ADP plus malate. The increased oxygen evolution observed when malate plus ADP was added with oxaloacetate was accompanied by pyruvate production. These results are consistent with oxaloacetate being decarboxylated via PEP carboxykinase. We suggest that the ATP required for oxaloacetate decarboxylation via PEP carboxykinase may be derived by phosphorylation coupled to malate oxidation in mitochondria. These bundle sheath cells apparently contain diffusion paths for the rapid transfer of compounds as large as adenine nucleotides.     

The kinetic mechanism of yeast phosphoenolpyruvate carboxykinase

The kinetic mechanism of yeast phosphoenolpyruvate carboxykinase, in the physiological direction, has been determined. Product inhibition using KHCO₃ showed competitive inhibition, when both oxalacetate (OAA) and ATP were varied. Phosphoenolpyruvate showed noncompetitive inhibition against OAA, and competitive inhibition with respect to ATP. Conversely, ADP showed competitive inhibition against OAA and noncompetitive inhibition vs. ATP. Dead-end inhibition studies with β-sulfopyruvate showed competitive inhibition against OAA and noncompetitive inhibition vs. ATP. Ethene-ATP exhibited competitive inhibition against ATP and noncompetitive inhibition with respect to OAA. These results are consistent with a random Bi-Ter mechanism with the formation of two abortive complexes: enzyme-ATP-ADP and enzyme-OAA-PEP.     

Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase. Mutagenesis at metal site 1

Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate (OAA) and ATP from PEP, ADP and CO(2). Mutations of PEP carboxykinase have been constructed where the residues His(225) and Asp(263), two residues of the enzyme's putative Mn(2+) binding site, were altered. Kinetic studies of the His225Glu, and Asp263Glu PEP carboxykinases show 600- and 16,800-fold reductions in V(max) relative to the wild-type enzyme, respectively, with minor alterations in K(m) for Mn(2+). Molecular modeling of wild-type and mutant enzymes suggests that the lower catalytic efficiency of the Asp263Glu enzyme could be explained by a movement of the lateral chain of Lys(248), a critical catalytic residue, away from the reaction center. The effect on catalysis of introducing a negatively charged oxygen atom in place of N(epsilon-2) at position 225 is discussed in terms of altered binding energy of the intermediate enolpyruvate.     

Regulation of phospho(enol)-pyruvate-and oxaloacetate-converting enzymes in Corynebacterium glutamicum

The presence and properties of the enzymes involved in the synthesis and conversion of phospho(enol)pyruvate (PEP) and oxaloacetate (OAA), the precursors for aspartate-derived amino acids, were investigated in three different Corynebacterium strains. This study revealed the presence of both PEP carboxykinase 0.29 mol·min^–1·mg^–1 of protein [units (U)·mg^–1] and PEP synthetase (0.13 U·mg^–1) in C. 2 glutamicum as well as pyruvate kinase (1.4 U·mg^–1) and PEP carboxylase (0.16 U·mg^–1). With the exception of PEP carboxykinase these activities were also present in glucose-grown C. flavum and C. lactofermentum. Pyruvate carboxylase activity was not detected in all three species cultivated on glucose or lactate. At least five enzyme activities that utilize OAA as a substrate were detected in crude extracts of C. glutamicum: citrate synthase (2 U·mg^–1), malate dehydrogenase (2.5 U·mg^–1), glutamate: OAA transaminase (1 U·mg^–1), OAA-decarboxylating activity (0.89 U·mg^–1) and the previously mentioned PEP carboxykinase (0.29 U·mg^–1). The partially purified OAA-decarboxylase activity of C. glutamicum was completely dependent on the presence of inosine diphosphate and Mn²⁺, had a Michaelis constant (K _m) of 2.0mm for OAA and was inhibited by ADP and coenzyme A (CoA). Examination of the kinetic properties showed that adenine nucleotides and CoA derivatives have reciprocal but reinforcing effects on the enzymes catalyzing the interconversion of pyruvate, PEP and OAA in C. glutamicum. A model for the regulation of the carbon flow based on these findings is presented.Correspondence to: M. S. M. Jetten     

Effect of a constant supply of different nitrogen sources on protein and carbohydrate content and enzyme activities of Anabaena variabilis cells

The effect of the nitrogen source on carbohydrate and protein contents and on several enzymatic activities involved in the carbon and nitrogen metabolism was studied in Anabaena variabilis ATCC 29413 cells grown under a constant supply of either N, NO⁻₃ or NH⁺₄ at different concentrations. An enhancement of protein content accompanied by a parallel decrease of carbohydrates was observed with increasing NO⁻₃ or NH⁺₄ concentrations in the medium. In cultures containing 0.1 m M NO⁻₃ or 0.1 m M NH⁺₄ nitrogenase (EC 1.18.6.1) activity was 74 and 66%, respectively, of that found in N₂-grown cells. This activity was still present with 1 m M NO⁻₃ or 1 m M NH⁺₄ in the medium and even with 10 m M NO⁻₃, but it was completely inhibited by 5 m M NH⁺₄. Ferredoxin-nitrate reductase (EC 1.7.7.2) activity was detected only in NO⁻₃ grown cells and simultaneously with nitrogenase activity. Increasing concentrations of combined nitrogen in the medium, especially NH⁺₄, promoted a concomitant decline of glutamine synthetase (EC 6.3.1.2), NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42), and NAD⁺-malate dehydrogenase (EC 1.1.1.37) activities, suggesting that these enzymes play an important role in the regulation of carbon-nitrogen metabolism in cyanobacteria.     

Carbonic anhydrases in higher plants and aquatic microorganisms

At physiological pH-values CO₂ and HCO⁻₃are the dominant inorganic carbon species and the interconversion between both is catalyzed by carbonic anhydrase (EC 4.2.1.1). This enzyme is widely distributed among photosynthetic organisms. In the first part of the review, the similarities and the differences of carbonic anhydrases from plants and animals are briefly described. In the second part recent advances in molecular biology to understand the structure of carbonic anhydrase from higher terrestrial plants as well as its involvement in photosynthetic CO₂ fixation are summarized. Lastly, the review deals with the presence of carbonic anhydrase in aquatic organisms including cyanobacteria, microalgae, macroalgae and angiosperms. Evidence for the presence of extracellular and intracellular isozymes in these organisms are discussed. The properties and function(s) of carbonic anhydrase during the operation of the inorganic carbon concentrating mechanism are also described.     

Regulation of nitrate uptake into Chara corallina cells via NH+4 stimulation of NO−3 efflux

Abstract. Net NO₃ uptake by NO⁻₃ deficient Chara cells was used to calculate [NO⁻₃]_c assuming that the cytoplasm occupies 10% total volume and that nitrate reduction and storage are negligible (i.e. maximum [NO⁻₃]_c was calculated). A linear relationship was found between NO⁻₃ efflux and [NO⁻₃]_c. There was an initial burst of NO⁻₃ efflux when NH⁺₄ was added, followed by a slower efflux rate which matched influx rate such that net NO⁻₃ uptake was zero. Over 50% of NO⁻₃ that had been taken up in 2 h was lost within the first 5 min of NH⁺₄ addition. The Nernst equation was used to predict the direction of the electrochemical driving force for NO⁻₃ entry. Under the experimental conditions used NO⁻₃ efflux is actively transported. The differential involvement of both NO⁻₃ influx and NO⁻₃ efflux in the regulation of NO⁻₃ uptake is discussed and a model is proposed to account for these results which envisages discrete NO⁻₃ influx and NO⁻₃ efflux carriers.