Purified Phosphoenolpyruvate Carboxylase Research Articles

Regulation of phospho enolpyruvate carboxylase from Crassula argentea: Effect of incubation with ligands and dilution on oligomeric state, activity, and allosteric properties

The relationship between the aggregation state and allosteric properties of purified phospho enolpyruvate carboxylase from Crassula argentea was examined using both kinetic and physical techniques. Analysis by native polyacrylamide gel electrophoresis showed that dilution induced a dissociation of the active tetramer to a less active dimer. Kinetic assays showed that inhibition of phospho enolpyruvate carboxylase by 5 m m malate measured at a saturating phospho enolpyruvate concentration rose to nearly 80% with increasing preassay dilution while the activity in the absence of malate remained constant. Kinetic bursts were observed when enzyme-initiated assays were measured at a subsaturating phospho enolpyruvate concentration. At saturating phospho enolpyruvate concentrations, however, increasing lags developed in response to increasing the preassay dilution of the enzyme. Further, dynamic laser-light scattering measurements showed that preincubation of the dilute enzyme with phospho enolpyruvate stabilized the tetramer while the presence of malate induced dimer formation. These observations confirm and extend earlier work with the extracted active malate insensitive night and less active, malate-sensitive day forms of the enzyme (Wu and Wedding [1985] Plant Physiol. 77, 667–675). Activity measured at subsaturating phospho enolpyruvate concentrations dropped with increasing preassay dilution of enzyme, while activation by 3.2 m m glucose 6-phosphate, assayed at a low phospho enolpyruvate concentration (0.044 m m), increased with dilution to nearly 400%. In this case activation results from a decrease in the control rate as the activity measured in the presence of glucose 6-phosphate was nearly constant, similar in effect to saturating phospho enolpyruvate in the assay. Glucose 6-phosphate induced tetramer formation of the dilute enzyme as measured by light-scattering similar to the effects induced by PEP. In addition, when diluted (dimeric) PEPC was preincubated with PEP or glucose 6-phosphate the enzyme became less sensitive to malate inhibition, while the active-site directed ligand 2-phosphoglycolate had no effect on malate inhibition. These results indicate that both the substrate PEP and the activator glucose 6-phosphate stabilize the active tetramer via binding and interaction at an activator site separate from the active site.

The influence of pH on substrate form specificity of phospho enolpyruvate carboxylase purified from Crassula argentea

Purified phospho enolpyruvate carboxylase from both the crassulacean acid metabolism plant Crassula argentea and the C 4 plant Zea mays was shown by kinetic studies at saturating fixed-varying concentrations of free Mg 2+ to selectively use the metal-complexed form of phospho enolpyruvate when assayed at pH 8.0. A similar response to added magnesium at high free phospho enolpyruvate concentrations was obtained for both enzymes, consistent with the use of the complex as the substrate. Kinetic studies at pH 7.0 indicated that at this pH the total concentration of phospho enolpyruvate (including both free and metal-complexed forms) could be used by the enzyme from C. argentea while the C 4 enzyme still utilized the complex. The loss of specificity induced by the decrease in the pH of the assay medium was accompanied by a decrease in the K m of this enzyme for phospho enolpyruvate whatever the form considered and an increase in V max K m . In contrast, a similar decrease of pH led to an increased K m of the C 4 enzyme for phospho enolpyruvate and a decrease of V max K m . For the enzyme from C. argentea (previously shown to contain an essential arginine at the active site), protection of activity by the different forms of substrate against inactivation by the specific arginyl reagent 2,3-butanedione changes markedly with pH. At pH 8.1, the metal complex is the better protector while at pH 7.0 free phospho enolpyruvate gives the best protection consistent with the observed kinetic changes in substrate form utilization. The relationship between the enzyme affinity for substrate, substrate specificity, and the requirement for magnesium for substrate turnover is discussed.

Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase.

A method was developed for the purification of phosphoenolpyruvate carboxylase from darkened maize leaves so that the enzyme retained its sensitivity to inhibition by malate. The procedure depended on the prevention of proteolysis by the inclusion of chymostatin in the buffers used during the purification. The purified enzyme was indistinguishable from that in crude extracts as judged by native polyacrylamide-gel electrophoresis. SDS/polyacrylamide-gel electrophoresis followed by immunoblotting, and Superose 6 gel filtration. Gel-filtration studies showed that the purified enzyme and the enzyme in extracts of darkened or illuminated leaves showed a concentration-dependent dissociation of tetrameric into dimeric forms. Purified phosphoenolpyruvate carboxylase and enzyme in crude extracts from darkened leaves were equally sensitive to inhibition by malate (Ki approx. 0.30 mM) under conditions where it existed in the tetrameric or dimeric forms, but the enzyme in crude extracts from illuminated leaves was less sensitive to malate inhibition (Ki approx. 0.95 mM) whether it was present as a tetramer or as a dimer. It is concluded that changes in the oligomerization state of phosphoenolpyruvate carboxylase are not directly involved in its regulation by light.

A kinetic study of the effects of phosphate and organic phosphates on the activity of phospho enolpyruvate carboxylase from Crassula argentea

The effects of phosphate and several phosphate-containing compounds on the activity of purified phospho enolpyruvate carboxylase (PEPC) from the crassulacean acid metabolism plant, Crassula argentea, were investigated. When assayed at subsaturating phospho enolpyruvate (PEP) concentrations, low concentrations of most of the compounds tested were found to stimulate PEPC activity. This activation, variable in extent, was found in all cases to be competitive with glucose 6-phosphate (Glc-6-P) stimulation, suggesting that these effectors bind to the Glc-6-P site. At higher concentrations, depending upon the effector molecule studied, deactivation, inhibition, or no response was observed. More detailed studies were performed with Glc-6-P, AMP, phosphoglycolate, and phosphate. AMP had previously been shown to be a specific ligand for the Glc-6-P site. The main effect of Glc-6-P and AMP on the kinetic parameters was to decrease the apparent K m and increase V max K m . AMP also caused a decrease in the V max of the reaction. In contrast, phosphoglycolate acted essentially as a competitive inhibitor increasing the apparent K m for PEP and decreasing V max K m . Inorganic phosphate had a biphasic effect on the kinetic parameters, resulting in a transient decrease in K m followed by an increase of the apparent K m for PEP with increasing concentration of phosphate. The V max also was decreased with increasing phosphate concentrations. Further, the enzyme appeared to respond to the complex of phosphate with magnesium. In the presence of a saturating concentration of AMP, no activation but rather inhibition was observed with increasing phosphate concentration. This is consistent with the binding of phosphate to two separate sites—the Glc-6-P activation site and an inhibitory site, a phenomenon that may be occurring with other phosphate containing compounds. High concentrations of phosphate with magnesium were found to protect enzyme activity when PEPC, previously shown to contain an essential arginine at the active site, was incubated with the specific arginyl reagent 2,3-butanedione, consistent with the binding of phosphate at the active site. Data were successfully fitted to a rapid equilibrium model allowing for binding of the phosphate-magnesium complex to both the activation site and the active site which accounts for the activation/deactivation observed at low substrate concentrations. Effects on the V max of the reaction are also addressed. Factors controlling the differential affinity of various effectors to the active site or activation site appear to include charge distribution, size, and other steric factors.

Regulatory seryl-phosphorylation of C 4 phospho enolpyruvate carboxylase by a soluble protein kinase from maize leaves

A reconstituted system composed of purified phospho enolpyruvate carboxylase (PEP-Case) and a soluble protein kinase (PK) from green maize leaves was developed to critically assess the effects of in vitro protein phosphorylation on the catalytic and regulatory (malate sensitivity) properties of the target enzyme. The PK was partially purified from light-adapted leaf tissue by ammonium sulfate fractionation (0–60% saturation fraction) of a crude extract and blue dextran-agarose affinity chromatography. The resulting preparation was free of PEPCase. This partially purified protein kinase activated PEP-Case from dark-adapted green maize leaves in an ATP-, Mg 2+-, time-, and temperature-dependent fashion. Concomitant with these changes in PEPCase activity was a marked decrease in the target enzyme's sensitivity to feedback inhibition by l-malate. The PK-mediated incorporation of 32P from [γ- 32P]ATP into the protein substrate was directly correlated with these changes in PEPCase activity and malate sensitivity. The maximal molar 32P-incorporation value was about 0.25 per 100-kDa PEPCase subunit (i.e., 1 per holoenzyme). Phosphoamino acid analysis of the 32P-labeled target enzyme by two-dimensional thin-layer electrophoresis revealed the exclusive presence of phosphoserine. These in vitro results, together with our recent studies on the light-induced changes in phosphorylation status of green maize leaf PEPCase in vivo ( J.-A. Jiao and R. Chollet (1988) Arch. Biochem. Biophys. 261, 409–417 ), collectively provide the first unequivocal evidence that the seryl-phosphorylation of the dark-form enzyme by a soluble protein kinase is responsible for the changes in catalytic activity and malate sensitivity of C 4 PEPCase observed in vivo during dark/light transitions of the parent leaf tissue.

The Effect of Adenine Nucleotides on Purified Phosphoenolpyruvate Carboxylase from the CAM Plant Crassula argentea

The effects of adenine nucleotides on phosphoenolypyruvate carboxylase were investigated using purified enzyme from the CAM plant, Crassula argentea. At 1 millimolar total concentration and with limiting phosphoenolpyruvate, AMP had a stimulatory effect, lowering the K(m) for phosphoenolpyruvate, ADP caused less stimulation, and ATP decreased the activity by increasing the K(m) for phosphoenolpyruvate. Activation by AMP was not additive to the stimulation by glucose 6-phosphate. Furthermore, AMP increased the K(a) for glucose 6-phosphate. Inhibition by ATP was competitive with phosphoenolpyruvate. In support of the kinetic data, fluorescence binding studies indicated that ATP had a stronger effect than AMP on phosphoenolpyruvate binding, while AMP was more efficient in reducing glucose 6-phosphate binding. As free Mg(2+) was held constant and saturating, these effects cannot be ascribed to Mg(2+) chelation. Accordingly, the enzyme response to the adenylate energy charge was basically of the "R" type (involving enzymes of ATP regenerating sequences) according to D. E. Atkinson's (1968 Biochemistry 7: 4030-4034) concept of energy charge regulation. The effect of energy charge was abolished by 1 millimolar glucose 6-phosphate. Levels of glucose 6-phosphate and of other putative regulatory compounds of phosphoenolpyruvate carboxylase were determined in total leaf extracts during a day-night cycle. The level of glucose 6-phosphate rose at night and dropped sharply during the day. Such a decrease in glucose 6-phosphate concentration could permit an increased control of phosphoenolpyruvate carboxylase by energy charge during the day.

Preparation and characterization of monoclonal antibodies against phosphoenolpyruvate carboxylase of Escherichia coli.

Twelve hybridoma clones which secrete monoclonal antibodies (mAb) against purified phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 were obtained. These 12 mAb were prepared from the ascites fluids of mice. Six among the 12 mAb formed precipitin lines with the enzyme on immunodiffusion. Four mAb inhibited the activity of the enzyme and 2 mAb enhanced it. Four mAb altered the sensitivity of the enzyme to allosteric effectors. Competitive enzyme-binding experiments among the 12 different mAb were also performed. The results showed that the 12 mAb can be classified into at least 8 groups.

Synthesis, storage, and stability of [4- 14C]oxaloacetic acid

A simple procedure for preparing [4- 14C]oxaloacetic acid based on the reaction between [ 14C]HCO 3 − and phosphoenolpyruvate catalyzed by phosphoenolpyruvate carboxylase is described. A simple method for preparing highly purified phosphoenolpyruvate carboxylase from maize leaves is described and the degradation of oxaloacetate under conditions of varying pH and divalent metal ion concentration is reported on. [4- 14C]Oxaloacetic acid is stable for several months in 0.1 m HCl solution at −80°C.

A comparison of the kinetic properties of phosphoenolpyruvate carboxylase from guard-cell and mesophyll-cell protoplasts of Commelina communis

Some kinetic properties of partially purified phosphoenolpyruvate carboxylase (PEPCase) from guard-cell and mesophyll-cell protoplasts of Commelina communis are described. The PEPCase activity inherent to each cell type was determined and the apparent K m (phosphoenolpyruvate) and K i (malate) were compared. Malate sensitivity was much higher (K i malate 0.4 mol m(-3)) in the extract of guard-cell protoplasts than in that of mesophyllcell protoplasts (K i malate 4.2 mol m(-3)). The stimulation of activity by glucose-6-phosphate in the presence of malate ('deinhibition') was also investigated in extracts from both cell types and was found to be similar to previously reported results with epidermal tissue. The effect of contamination of an extract of guard-cell protoplasts with mesophyll-cell protoplasts was measured in the presence and absence of malate. It was found that a small amount to mesophyll-cell contaminant appears to desensitize the malate inhibition of PEPCase from guard-cell protoplasts. It is concluded that experiments which use epidermal tissue to study guardcell PEPCase may give misleading information as a consequence of mesophyll contamination.

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.

Recovery of active, highly purified phosphoenolpyruvate carboxylase from specific immunoadsorbent column

FEBS LettersVolume 118, Issue 1 p. 31-34 Full-length articleFree Access Recovery of active, highly purified phosphoenolpyruvate carboxylase from specific immunoadsorbent column Jean Vidal, Jean Vidal Laboratoire de Biologie Végétale, ERA CNRS no. 799, 54 037-Nancy Cedex, FranceSearch for more papers by this authorGuy Godbillon, Guy Godbillon Laboratoire de Biologie Animale, Université de Nancy I, Case Officielle no. 140, 54 037-Nancy Cedex, FranceSearch for more papers by this authorPierre Gadal, Pierre Gadal Laboratoire de Biologie Végétale, ERA CNRS no. 799, 54 037-Nancy Cedex, FranceSearch for more papers by this author Jean Vidal, Jean Vidal Laboratoire de Biologie Végétale, ERA CNRS no. 799, 54 037-Nancy Cedex, FranceSearch for more papers by this authorGuy Godbillon, Guy Godbillon Laboratoire de Biologie Animale, Université de Nancy I, Case Officielle no. 140, 54 037-Nancy Cedex, FranceSearch for more papers by this authorPierre Gadal, Pierre Gadal Laboratoire de Biologie Végétale, ERA CNRS no. 799, 54 037-Nancy Cedex, FranceSearch for more papers by this author First published: August 25, 1980 https://doi.org/10.1016/0014-5793(80)81211-7Citations: 54AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 R.S. Bandurski, C.M. Greiner, J. Biol. Chem., 204, (1953), 781– 786. 2 H. Ozaki, I. Shiio, J. Biol. Chem., 66, (1969), 297– 311. 3 M.D. Hatch, C.R. Slack, Ann. Rev. Plant Physiol., 21, (1970), 141– 162. 4 B.G. Sutton, C.B. Osmond, Plant Physiol., 50, (1972), 360– 365. 5 C. Willmer, R. Kanai, J.E. Pallas jr, C.C. Black jr, Life Sci., 12, (1973), 151– 155. 6 L. Jacobson, Plant Physiol., 30, (1955), 264– 269. 7 K.J. Bonugli, D.D. Davies, Plant, 133, (1977), 281– 287. 8 O. Queiroz, C. Celati, C. Morel, Physiol. Veg., 10, (1972), 765– 781. 9 S.K. Mukerji, I.P. Ting, Arch. Biochem. Biophys., 143, (1971), 297– 317. 10 S.K. Mukerji, Arch. Biochem. Biophys., 182, (1977), 343– 351. 11 I.P. Ting, C.B. Osmond, Plant Physiol., 51, (1973), 448– 453. 12 J.H. Chen, R.F. Jones, Biochim. Biophys. Acta, 214, (1970), 318– 325. 13 J. Brulfert, M.C. Arrabaca, O. Guerrier, O. Queiroz, Planta, 146, (1979), 129– 133. 14 D.D. Davies, Ann. Rev. Plant Physiol., 30, (1979), 131– 158. 15 J. Vidal, G. Cavalié, Physiol. Veg., 12, (1974), 175– 188. 16 J. Vidal, G. Cavalié, P. Gadal, Plant Sci. Lett., 7, (1976), 265– 270. 17 M.D. Hatch, I.R. Oliver, Aust. J. Plant Physiol., 5, (1978), 571– 580. 18 O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem., 193, (1951), 265– 275. 19 R.K. Scopes, Anal. Biochem., 59, (1974), 277– 282. 20 K. Uedan, Plant Physiol., 57, (1976), 906– 910. 21 R. Jones, M.B. Wilkins, J.R. Coggins, C.A. Fewson, A.D.B. Malcolm, Biochem. J., 175, (1978), 391– 397. Citing Literature Volume118, Issue1August 25, 1980Pages 31-34 ReferencesRelatedInformation

Activation and Inactivation of Phosphoenolpyruvate Carboxylase in Leaf Extracts From C4 Species

The stability of phosphoenolpyruvate carboxylase (EC 4.1.1.31) was examined following extraction of the enzyme from leaves of several C4 plants. Extracts were rapidly processed on small Sephadex G-25 columns to free protein of small-molecular-weight compounds. With most of the species examined, activity was rapidly lost at both 0 and 25°C when the pH was about 7.8 or higher. Addition of bovine serum albumin to extracts incubated at 25°C and pH 8.2 not only prevented inactivation with several species, but resulted in a substantial increase in activity. The addition of dithiothreitol plus Mg2+ to extracts from some of these species reduced or prevented inactivation. With extracts maintained at 0°C, addition of either bovine serum albumin or dithiothreitol was effective only in reducing the rate of inactivation in extracts. Phosphoenolpyruvate carboxylase activity remained stable, or increased substantially, when extracts buffered between pH 7.4 and 6.9 were incubated at either 0 or 25°C. Activation was usually complete within an hour and was often significantly greater at 25°C or when bovine serum albumin was added. The activity of partially purified phosphoenolpyruvate carboxylase from Zea mays was similarly affected by pH, temperature, and bovine serum albumin. The present studies raise doubts about the accuracy of phosphoenolpyruvate carboxylase determinations made during the course of some previous studies on C4 species. Reliable procedures for the determination of phosphoenolpyruvate carboxylase activity in C4 plant extracts are described. Possible physiological implications of the results are considered.

Reduced nicotinamide adenine dinucleotide-activated phosphoenolpyruvate carboxylase in Pseudomonas MA: potential regulation between carbon assimilation and energy production

Comparison of enzyme activities in crude extracts of methylamine-grown Pseudomonas MA (ATCC 23319) to those in succinate-grown cells indicates the involvement of an acetyl coenzyme A-independent phosphoenolpyruvate carboxylase in one-carbon metabolism. The purified phosphoenolpyruvate carboxylase is activated specifically by reduced nicotinamide adenine dinucleotide (KA = 0.2 mM). The regulatory properties of this enzyme suggests that phosphoenolpyruvate serves as a focal point for both carbon assimilation and energy metabolism.

Malonate-inhibition of allosteric phosphoenolpyruvate carboxylase from Setaria italica

Summary Malonate inhibited phosphoenolpyruvate carboxylase activity in crude extracts of Setaria italica Beauv. and Pennisetum typhoides S & H leaves. Kinetic studies revealed the competetive inhibition of partially purified phosphoenolpyruvate carboxylase preparation from Setaria italica . Glucose-6-phosphate activated the enzyme. Oxalacetate also competetively inhibited the enzyme and malate in a noncompetetive manner. Results clearly indicated the allosteric nature of phosphoenolpyruvate carboxylase from Setaria italica .

Phosphoenolpyruvate Carboxylase: Activation by Nucleotides as a Possible Compensatory Feedback Effect

Abstract The activation of partially purified phosphoenolpyruvate carboxylase from Salmonella typhimurium by various pyrimidine and purine nucleotides is described. The most potent activators are found to be cytidine diphosphate, guanosine triphosphate, and cytidine monophosphate, in that order. It is shown that in the presence of acetyl coenzyme A the affinity of the nucleotides for their binding site is increased. Evidence is presented to demonstrate that the nucleotides do not participate in the enzyme reaction, but act only catalytically. From these studies the physiological concept of compensatory feedback loops in the control of branched pathways is presented.