Changes in phosphoenolpyruvate carboxylase and ribulosebisphosphate carboxylase activities in tobacco plants infected with tobacco mosaic virus

Considerable changes in the activities of phosphoenolpyruvate carboxylase and ribulosebisphosphate carboxylase were found inNicotiana tabacum cv. Sarasun plants infected with TMV. Ribulosebisphosphate carboxylase is inhibited at the time of maximum TMV reproduction, but its decreased activity is at the same time partly compensated by phosphoenol-pyruvate carboxylase in the shoots of infected plants. The pattern of activity of this enzyme nearly exactly reflects the pattern of reproduction of the tobacco mosaic virus.     

Short-term nitrogen-induced modulation of phosphoenolpyruvate carboxylase in tobacco and maize leaves

Untransformed maize and tobacco plants and tobacco plants constitutively expressing nitrate reductase were grown with sufficient NO(3)- to support maximal growth. Four days prior to treatment the tobacco plants were deprived of nitrogen. Excised maize leaves and tobacco leaf discs were fed with either 40 mM KNO(3) or 40 mM KCl (control) in the light. Phosphoenolpyruvate (PEP) carboxylase (Case) activity was measured at 0.3 mM and 3 mM PEP. The light- induced increase in PEPCase V(max) was greater in maize than tobacco. Furthermore light decreased malate sensitivity in maize (which was N-replete) but not in N-deficient tobacco. NO(3)- treatment increased PEPCase V:(max) values in both species and decreased the sensitivity to inhibition by malate, but effects of NO(3)- were much more pronounced in tobacco than maize. PEPCase kinase activity was, however, greater in maize leaves NO(3)- than in the Cl(-)-treated controls, suggesting that it is responsive to leaf nitrogen supply. A correlation between foliar glutamine content and PEPCase activity was observed. It is concluded that PEPCase is sensitive to N metabolites which favour increased flow through the anapleurotic pathway in both C(3) and C(4) plants.     

On the molecular mechanism of maize phosphoenolpyruvate carboxylase activation by thiol compounds

Incubation of purified phosphoenolpyruvate carboxylase from Zea mays L. leaves with dithiothreitol resulted in an almost 2-fold increase in the enzymic activity. The activated enzyme showed the same affinity for its substrates and the same sensitivity with respect to malate and oxalacetate inhibition. The activation induced by dithiothreitol was reversed by diamide, an oxidant of vicinal dithiols, suggesting that the redox state of disulfide bonds of the enzyme may be important in the expression of the maximal catalytic activity.

Titration of thiol groups before and after activation of maize phosphoenolpyruvate carboxylase by dithiothreitol shows an increase of the accessible groups from 8 to 12 suggesting that the reduction of two disulfide bonds accompanied the activation. The thiols exposed by the treatment with dithiothreitol were available to reagents in nondenatured enzyme and two of them were reoxidized to a disulfide bond by diamide. It is concluded that the mechanism of phosphoenolpyruvate carboxylase activation by dithiothreitol involves the net reduction of two disulfide bonds in the enzyme.

     

Sequence analysis of cDNA encoding phosphoenolpyruvate carboxylase from cultured tobacco cells

The complete nucleotide sequence of cDNA encoding phosphoenolpyruvate carboxylase (PEPCase) from cultured tobacco (a C₃ plant) cells was determined and the deduced amino acid sequence was compared with those of PEPCases from other higher plants.     

Inhibition of phosphoenolpyruvate carboxylase by malate

Malate has been noted to be a `mixed' inhibitor of phosphoenolpyruvate (PEP) carboxylase. The competitive portion of this inhibition appears to be fairly constant regardless of the condition of the enzyme being measured, but the noncompetitive (V-type) inhibition is subject to variation depending on the source of the enzyme, its storage condition, the presence or absence of various ligands, and differences in pH. In the case of the maize (Zea mays L.) phosphoenolpyruvate carboxylase (PEPC), the V-type inhibition by malate is much less pronounced at pH 8 than at pH 7. Examination of the response of the maize PEPC to PEP concentration reveals a pronounced cooperativity at pH 8 which is not present at pH 7, and which results in the disappearance of the V-type inhibition at pH 8. The ability of high concentrations of PEP to convert PEPC from a form readily inhibited by malate to one resistant to malate inhibition has been previously demonstrated and we attribute the cooperativity shown at pH 8 to this response to high levels of PEP. Support for this proposal is provided by studies of the enzyme at pH 7 and pH 8 run in 20% glycerol. In this case there was no V-type inhibition of PEPC at either pH. Treatment with 20% glycerol has been shown to result in the aggregation of maize PEPC.     

Inactivation of maize phosphoenolpyruvate carboxylase by urea

Phosphoenolpyruvate carboxylase purified from leaves of maize (Zea mays, L.) is sensitive to the presence of urea. Exposure to 2.5 m urea for 30 min completely inactivates the enzyme, whereas for a concentration of 1.5 m urea, about 1 h is required. Malate appears to have no effect on inactivation by urea of phosphoenolpyruvate carboxylase. However, the presence of 20 mm phosphoenolpyruvate or 20 mm glucose-6-phosphate prevents significant inactivation by 1.5 m urea for at least 1 h. The inactivation by urea is reversible by dilution. The inhibition by urea and the protective effects of phosphoenolpyruvate and glucose-6-phosphate are associated with changes in aggregation state.     

Hydrolysis of phosphoenolpyruvate catalyzed by phosphoenolpyruvate carboxylase from Zea mays.

In addition to the normal carboxylation reaction, phosphoenolpyruvate carboxylase from Zea mays catalyzes a HCO3(-)-dependent hydrolysis of phosphoenolpyruvate to pyruvate and Pi. Two independent methods were used to establish this reaction. First, the formation of pyruvate was coupled to lactate dehydrogenase in assay solutions containing high concentrations of L-glutamate and aspartate aminotransferase. Under these conditions, oxalacetic acid produced in the carboxylation reaction was efficiently transaminated, and decarboxylation to form spurious pyruvate was negligible. Second, sequential reduction of oxalacetate and pyruvate was achieved by initially running the reaction in the presence of malate dehydrogenase with NADH in excess over phosphoenolpyruvate. After the reaction was complete, lactate dehydrogenase was added, thus giving a measure of pyruvate concentration. At pH 8.0 in the presence of Mg2+, the rate of phosphoenolpyruvate hydrolysis was 3-7% of the total reaction rate. The hydrolysis reaction catalyzed by phosphoenolpyruvate carboxylase was strongly metal dependent, with rates decreasing in the order Ni2+ greater than Co2+ greater than Mn2+ greater than Mg2+ greater than Ca2+. These results suggest that the active site metal ion binds to the enolate oxygen, thus stabilizing the proposed enolate intermediate. The more stable the enolate, the less reactive it is toward carboxylation and the greater the opportunity for hydrolysis.     

Role of cysteine in activation and allosteric regulation of maize phosphoenolpyruvate carboxylase

The effect of 5-5′-dithiobis-2-nitrobenzoate (DTNB) on the kinetic parameters and structure of phosphoenolpyruvate carboxylase purified from maize (Zea mays L.) has been studied. The V_max is found to be independent of the presence of this thiol reagent. The K_m is increased upon oxidation of cysteines by DTNB. At a substrate concentration higher than K_m (3.1 millimolar Mgphosphoenolpyruvate), a significant reversible decrease of the activity is observed. Malate has little effect in preventing the modification of these cysteines. The V type inhibition by malate was also studied at a saturating phosphoenolpyruvate level (9.3 millimolar Mgphosphoenolpyruvate). In the presence of 50 micromolar DTNB, up to 60% inhibition is caused by 15 millimolar malate; however, in the presence of both 50 micromolar DTNB and 50 millimolar dithiothreitol (DTT) this inhibition is reduced to 20%. The presence of DTT alone increases the size of the phosphoenolpyruvate carboxylase molecule as determined by light scattering. The activity at nonsaturating substrate concentration is increased by 36% in the presence of DTT. The oligomerization equilibrium between the dimer and the tetrameric form of the enzyme is affected by cysteine. The K_m for the substrate, the sensitivity toward malate, and the size of the enzyme are found to be modified upon incubation in the presence of DTT.     

Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagentK

Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg²⁺ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.     

Regulation of phosphoenolpyruvate carboxylase of Zea mays by metabolites

Crude preparations of phosphoenolpyruvate carboxylase obtained from aetiolated seedlings of Zea mays are unstable but can be stabilized with glycerol. At the pH optimum of 8.3, the K(m) value for phosphoenolpyruvate is 80mum. When assayed at 30 degrees C, the enzyme shows normal Michaelis-Menten kinetics, but when assayed at 45 degrees C sigmoid kinetics are exhibited. At pH7.0 the enzyme is inhibited by a number of dicarboxylic acids and by glutamate and aspartate. d and l forms of the hydroxy acids and amino acids are inhibitory and the kinetics approximate to simple non-competitive inhibition. The same compounds produce less inhibition at pH7.6 than at pH7.0 and the kinetics of inhibition are more complex. The enzyme is activated by P(i), by SO(4) (2-) and by a number of sugar phosphates. Maximum activation occurs at acid pH values, where enzyme activity is lowest. The enzyme is activated by AMP and inhibited by ADP and ATP so that the response to energy charge is of the R type and is thus at variance with Atkinson's (1968) concept of energy charge. The physiological significance of the response to metabolites is discussed.     

Inhibition of phosphoenolpyruvate carboxylase from maize by 2-phosphoglycollate

The phosphoenolpyruvate carboxylase from maize leaf was strongly inhibited by 2-phosphoglycollate. The pH of the reaction did not influence the extent of inhibition by 2-phosphoglycollate. The kinetic analysis of the inhibition data by Lineweaver-Burk method showed that 2-phosphoglycollate inhibition was competitive with respect to phosphoenolpyruvate. The secondary plot of the data showed nonlinearity indicating that there may be two 2-phosphoglycollate binding sites with Ki values of 0.4 mM and 0.16 mM. The biphasic nature of the inhibition was also evident when the data were plotted using the method of Dixon. 2-phosphoglycollate inhibition was uncompetitive with respect to Mg²⁺ suggestting that it binds only to enzyme-Mg²⁺ complex.     

Inhibition of maize leaf phosphoenolpyruvate carboxylase by diethyl oxaloacetate

Diethyl oxaloacetate was found to be a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase activity with respect to the substrate phosphoenolpyruvate. The K_i values, based on total diethyl oxaloacetate, decreased with increasing pH, while the K_i values, based on the enol tautomer (average of 4 M), were similar and independent of pH. The results suggest that inhibition is dependent on the enol tautomer. Diethyl oxaloacetate was a weak inhibitor following treatment of the enzyme with dithiothreitol; inhibition could be restored by treatment with diamide, indicating inhibition depends on the reduction state of thiol groups on the enzyme.Abbreviations DTT dithiothreitol - HPLC high performance liquid chromatography - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - Tricine N-tris(hydroxymethyl)methylglycine     

Metabolite activation of crassulacean Acid metabolism and c(4) phosphoenolpyruvate carboxylase

The effects of glycine, alanine, serine, and various phosphorylated metabolites on the activity of phosphoenolpyruvate (PEP) carboxylase from Zea mays and Crassula argentea were studied. The maize enzyme was found to be activated by amino acids at a site that is separate from the glucose 6-phosphate binding site. The combination of glycine and glucose 6-phosphate synergistically reduced the apparent K_m of the enzyme for PEP and increased the apparent V_max. Of the amino acids tested, glycine showed the lowest apparent K_a and caused the greatest activation. d-Isomers of alanine and serine were more effective activators than the l-isomers. Unlike the maize enzyme, the Crassula enzyme was not activated by amino acids. Activation of either the Crassula or maize enzyme by glucose 6-phosphate occurred without dephosphorylation of the activator molecule. Furthermore, the Crassula enzyme was activated by two compounds containing phosphonate groups whose carbon-phosphorus bonds were not cleaved by the enzyme. A study of analogs of glucose 6-phosphate with Crassula PEP carboxylase revealed that the identity of the ring heteroatom was a significant structural feature affecting activation. Activation was not highly sensitive to the orientation of the hydroxyl group at the second or fourth carbon positions or to the presence of a hydroxyl group at the second position. However, the position of the phosphate group was found to be a significant factor.     

Expression of a phosphoenolpyruvate carboxylase promoter from Mesembryanthemum crystallinum is not salt-inducible in mature transgenic tobacco

The 5 flanking region of a salt-stress-inducible, CAM-specific phosphoenolpyruvate carboxylase (PEPC) gene from the facultative halophyte Mesembryanthemum crystallinum, was fused to the -glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum SR1. The Ppc1 promoter displayed high levels of expression in transgenic tobacco quantitatively and qualitatively similar to a full-length 35S CaMV-GUS construct. Histochemical assays revealed that the full-length Ppc1-GUS fusions expressed GUS activity in all tissues except in root tips. While tobacco is capable of utilizing the Ppc1 cis-acting regulatory regions from M. crystallinum to yield high levels of constitutive expression, this glycophyte fails to direct a stress-inducible pattern of gene expression typical of this promoter in its native, facultative halophytic host.     

Interference by phosphatases in the spectrophotometric assay for phosphoenolpyruvate carboxylase

The aim of this work was to discover the extent of interference by phosphoenolpyruvate (PEP) phosphatase in spectrophotometric assays of PEP carboxylase (EC 4.1.1.31) in crude extracts of plant organs. The presence of PEP phosphatase and lactate dehydrogenase (EC 1.1.1.27) in extracts leads to PEP-dependent NADH oxidation that is independent of PEP carboxylase activity, and hence to overestimation of PEP carboxylase activity. In extracts of three organs of pea (Pisum sativum L.: leaves, developing embryos, and Rhizobium nodules), two organs of wheat (Triticum aestivum L.: developing grain and endosperm), and leaves of Moricandia arvensis (L.) D.C., lactate dehydrogenase activity was at most only 16% of that of PEP carboxylase at the pH optimum for PEP carboxylase activity. Endogenous PEP phosphatase and lactate dehydrogenase are thus unlikely to interfere seriously with the assay for PEP carboxylase at its optimum pH. Addition of lactate dehydrogenase to PEP carboxylase assays— a proposed means of correcting for nonenzymic decarboxylation of oxaloacetate to pyruvate—resulted in increases in PEP-dependent NADH oxidation from zero (Rhizobium nodules) to 131% (wheat grains). There was no obvious relationship between the magnitude of this increase and conditions in the assay that might promote oxaloacetate decarboxylation. However, the magnitude of the increase was highly positively correlated with the activity of PEP phosphatase in the extract. Addition of lactate dehydrogenase to PEP carboxylase assays can thus result in very large overestimations of PEP carboxylase activity, and should only be used as a means of correction for oxaloacetate decarboxylation for extracts with negligible PEP phosphatase activity.     

Regulation at the phosphoenolpyruvate branchpoint in Azotobacter vinelandii: phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Azotobacter vinelandii, like the corresponding enzyme from other organisms, is activated by acetyl coenzyme A and inhibited by l-aspartate. Both modifiers affect primarily the affinity of the enzyme for phosphoenolpyruvate. This is the first enzyme with a strictly anaplerotic (intermediate-replacing) function to be tested for response to the adenylate energy charge; it is entirely insensitive to variation in charge. The results suggest that carboxylation of phosphoenolpyruvate in this organism is controlled by negative feedback from aspartate and by the stimulatory effect of acetyl coenzyme A. The adenylate energy charge may be expected to affect the rate of this reaction indirectly through its effects on the concentrations of acetyl coenzyme A and l-aspartate.