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PubMed1986

Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase.

The stereochemistry of the carboxylation of phosphoenolpyruvate to yield oxalacetate, catalyzed by chicken liver phosphoenolpyruvate carboxykinase and by Ascaris muscle phosphoenolpyruvate carboxykinase, was determined. The substrate (Z)-3-fluorophosphoenolpyruvate was used for the stereochemical analysis. The carboxylation reaction was coupled to malate dehydrogenase to yield 3-fluoromalate, and the stereochemistry of the products was identified by 19F NMR. In separate experiments, the enantiomeric tautomers of 3-fluorooxalacetate were shown to be utilized by malate dehydrogenase to yield (2R,3R)- and (2R,3S)-3-fluoromalate in nearly identical amounts. The products were identified by 19F NMR. When (Z)-3-fluorophosphoenolpyruvate was used as a substrate for phosphoenolpyruvate carboxykinase from avian liver and from Ascaris, and malate dehydrogenase was used to trap the product, only a single diastereomer was observed. This product was shown to be (2R,3R)-3-fluoromalate in each case. The assignments were based on coupling constants taken from Keck et al. [Keck, R., Hess, H., & Rétey, J. (1980) FEBS Lett. 114, 287]. These results indicate that the stereochemistry of carboxylation, catalyzed by chicken phosphoenolpyruvate carboxykinase and by Ascaris phosphoenolpyruvate carboxykinase, is identical and takes place from the si side of the enzyme-bound phosphoenolpyruvate. The carboxylation reaction was run both in H2O and in D2O. No deuterium incorporation into fluoromalate was shown to occur. The product 3-fluorooxalacetate is thus released from phosphoenolpyruvate carboxykinase as the keto form and is reduced more rapidly by reduced nicotinamide adenine dinucleotide with malate dehydrogenase than by the occurrence of tautomerization.

Animals↗

PubMed1978

Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates.

1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13mum produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the K(m) values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn(2+) in addition to Mg(2+) for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.

Alanine↗

PubMed1993

Alteration of growth yield by overexpression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in Escherichia coli.

Phosphoenolpyruvate and oxaloacetate are key intermediates at the junction between catabolism and biosynthesis. Alteration of carbon flow at these branch points will affect the growth yield and the formation of products. We attempted to modulate the metabolic flow between phosphoenolpyruvate and oxaloacetate by overexpressing phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase from a multicopy plasmid under the control of the tac promoter. It was found that overexpression of phosphoenolpyruvate carboxylase decreased the rates of glucose consumption and organic acid excretion, but the growth and respiration rates remained unchanged. Consequently, the growth yield on glucose was improved. This result indicates that the wild-type level of phosphoenolpyruvate carboxylase is not optimal for the most efficient glucose utilization in batch cultures. On the other hand, overexpression of phosphoenolpyruvate carboxykinase increased glucose consumption and decreased oxygen consumption relative to those levels required for growth. Therefore, the growth yield on glucose was reduced because of a higher rate of fermentation product excretion. These data provide useful insights into the regulation of central metabolism and facilitate further manipulation of pathways for metabolite production.

Escherichia coli↗

PubMed1984

Stereoselectivity of interaction of phosphoenolpyruvate analogues with various phosphoenolpyruvate-utilizing enzymes.

The halogenated phosphoenolpyruvate analogues (Z)-phosphoenol-3-fluoropyruvate, (E)-phosphoenol-3-fluoropyruvate, and (Z)-phosphoenol-3-bromopyruvate were synthesized and purified. The analogues were characterized by 1H and by 19F NMR where applicable. Absolute stereoselectivity of the fluorophosphoenolpyruvate isomers as substrates with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate phosphate dikinase was observed. The Z isomer exhibited substrate activity with these enzymes while no substrate activity was measured with the E isomer. Both isomers exhibited substrate activity with the enzyme pyruvate kinase, however, with a substantial decrease in the Vmax/Km ratio compared to phosphoenolpyruvate as the substrate. A metal ion dependent stereoselectivity of inhibition was measured for these analogues with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate kinase. The cation activator appears to affect the specificity and thus the catalytic site of these enzymes. Proton longitudinal relaxation rate titrations demonstrate that the dissociation constants, K3, of the fluorophosphoenolpyruvate isomers from the enzyme-Mn complex agree, in most cases, with the measured KI values and analogue binding resembles phosphoenolpyruvate binding. With the enzyme phosphoenolpyruvate carboxykinase, the KI not equal to K3 for (E)-fluorophosphoenolpyruvate which suggests that the binding of the E isomer is affected by the presence of the other substrates. The halogenated derivatives apparently undergo an enzyme-Mn catalyzed Michael-type addition reaction with the bromo-substituted analogue decomposing much faster than the fluoro analogues.

Animals↗

PubMed1992

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.

Bicarbonates↗

PubMed1988

Identification of substrate and effector binding sites of phosphoenolpyruvate carboxylase from Crassula argentea. A possible role of phosphoenolpyruvate as substrate and activator.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) purified from leaves of the crassulacean acid metabolism plant (Crassula argentea) was chemically modified by the specific arginyl reagent 2,3-butanedione. Modification resulted in enzyme inactivation which followed pseudo first-order kinetics. Participation of arginyl residues involved in the binding of or response to both phosphoenolpyruvate and malate, respectively, was established. Inactivation and protection studies suggest the presence of three sites involved in the binding of the substrate, phosphoenolpyruvate, the activator, glucose 6-phosphate, and the inhibitor, malate. Studies using both fluorescence measurements of binding and steady-state kinetic methods indicate that phosphoenolpyruvate can bind both to the active site and to the activator site. Evidence for stimulation of the activity of phosphoenolpyruvate carboxylase upon the binding of substrate to the activation site was provided by kinetic studies using AMP, previously shown to be a specific ligand for the activation site.

Carboxy-Lyases↗

PubMed1983

Facile oxygen exchanges of phosphoenolpyruvate and preparation of [18O]phosphoenolpyruvate.

Phosphoenolpyruvate when heated in acidic solution exchanges its phosphoryl and carboxyl oxygens rapidly and its enolic oxygen much more slowly with oxygens from water. The incorporation of 18O into phosphoenolpyruvate was measured by gas chromatography-mass spectrometry and phosphorus-31 nuclear magnetic resonance after heating in H218O at 98 degrees C. The rates of exchange of all six oxygens of phosphoenolpyruvate with water increase with increasing acidity, and the phosphoryl oxygens exchange more rapidly than the carboxyl oxygens. The rate of exchange of each oxygen of the phosphoryl group is 16-fold greater than the hydrolysis rate at 1 N HCl. This provides a simple and useful method for the synthesis of [18O]phosphoenolpyruvate highly enriched in its phosphoryl-group oxygens. An enrichment of 89% was obtained with a 50% yield. The [18O]-phosphoenolpyruvate showed a binomial distribution of 18O in the phosphoryl-group oxygens. The exchange may be explained by the reversible formation of a transient cyclic phosphate and, for exchange of the enolic oxygen, a transient acyl phosphate. Preparation of [18O]phosphoenolypyruvate from [18O]Pi by a chemical synthesis from beta-chlorolactate was not satisfactory because of drastic loss of 18O during the procedures used. Some loss of 18O also occurred during an enzymic synthesis with KCNO, [18O]Pi, carbamate kinase, and pyruvate kinase.

Gas Chromatography-Mass Spectrometry↗

PubMed2000

Purification and characterization of phosphoenolpyruvate carboxylase from Brassica napus (rapeseed) suspension cell cultures: implications for phosphoenolpyruvate carboxylase regulation during phosphate starvation, and the integration of glycolysis with nitrogen assimilation.

Phosphoenolpyruvate carboxylase (PEPC) specific activity increased by 250% following 8 to 10 days of Pi starvation of Brassica napus suspension cells. Densitometric scanning of PEPC immunoblots revealed a close correlation between PEPC activity and the amount of the antigenic 104-kDa PEPC subunit. To further assess the influence of Pi deprivation on PEPC, the enzyme was purified from Pi-sufficient (+Pi) and Pi-starved (-Pi) cells to electrophoretic homogeneity and final specific activities of 37-40 micromol phosphoenolpyruvate utilized per min per mg protein. Gel filtration, SDS/PAGE, and CNBr peptide mapping indicated that the +Pi and -Pi PEPCs are both homotetramers composed of an identical 104-kDa subunit. Respective pH-activity profiles, phosphoenolpyruvate saturation kinetics, and sensitivity to L-malate inhibition were also indistinguishable. Kinetic studies and phosphatase treatments revealed that PEPC of the +Pi and -Pi cells exists mainly in its dephosphorylated (L-malate sensitive) form. Thus, up-regulation of PEPC activity in -Pi cells appears to be solely due to the accumulation of the same PEPC isoform being expressed in +Pi cells. PEPC activity was modulated by several metabolites involved in carbon and nitrogen metabolism. At pH 7.3, marked activation by glucose 6-phosphate and inhibition by L-malate, L-aspartate, L-glutamate, DL-isocitrate, rutin and quercetin was observed. The following paper provides a model for the coordinate regulation of B. napus PEPC and cytosolic pyruvate kinase by allosteric effectors. L-Aspartate and L-glutamate appear to play a crucial role in the control of the phosphoenolpyruvate branchpoint in B. napus, particularly with respect to the integration of carbohydrate partitioning with the generation of carbon skeletons required during nitrogen assimilation.

Brassica↗

PubMed2005

Transient state kinetics of Enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system of Escherichia coli: equilibrium and second-order rate constants for the phosphotransfer reactions with phosphoenolpyruvate and HPr.

The first two reactions in the phosphotransfer sequence of bacterial phosphoenolpyruvate:glycose phosphotransferase systems are the autophosphorylation of Enzyme I by phosphoenolpyruvate followed by the transfer of the phospho group to the low-molecular weight protein, HPr. Transient state kinetic methods were used to estimate the second-order rate constants for both phosphotransfer reactions. These measurements support previous conclusions that only the dimer of Enzyme I, EI2, is autophosphorylated, and that the rate of formation of dimer is slow compared to the rate of its phosphorylation. The rate constants of the two autophosphorylation reactions of EI2 by PEP are 6.6 x 10(6) M(-1) s(-1), and differ from one another by a factor of less than 3. The rate constant for the transfer reaction between phospho-EI2 and HPr is unusually large for a covalent reaction between two proteins (220 x 10(6) M(-1) s(-1)), while the constant for the reverse reaction is 4.2 x 10(6) M(-1) s(-1). Using the previously reported equilibrium constant for the autophosphorylation reaction, 1.5, the overall equilibrium constant for phosphotransfer from PEP to HPr is 80, somewhat higher than that previously reported. The results also show that EI2 can phosphorylate multiple molecules of HPr without dissociating to a monomer (EI), and that EI can accept a phospho group from phospho-HPr. These results are directly applicable to predicting the rates of phosphoenolpyruvate phosphotransferase system sugar uptake in whole cells.

Bacterial Proteins↗

PubMed1971

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.

Aspartic Acid↗

PubMed1990

Kinetic evidence of the existence of a regulatory phosphoenolpyruvate binding site in maize leaf phosphoenolpyruvate carboxylase.

Phenylphosphate, a structural analog of phosphoenolpyruvate (PEP), was found to be an activator of phosphoenolpyruvate carboxylase (PEP carboxylase) purified from maize leaves. This finding suggested the presence in the enzyme of a regulatory site, to which PEP could bind. We carried out kinetic studies on this enzyme using controlled concentrations of free PEP and of Mg-PEP complex and developed a theoretical kinetic model of the reaction. In summary, the main conclusions drawn from our results, and taken as assumptions of the model, were the following: (i) The affinity of the active site for the complex Mg-PEP is much higher than that for free PEP and Mg2+ ions, and therefore it can be considered that the preferential substrate of the PEP-catalyzed reaction is Mg-PEP. (ii) The enzyme has a regulatory site specific for free PEP, to which Mg2+ ions can not bind. (iii) The binding of free PEP, or an analog molecule, to this regulatory site yields a modified enzyme that has much lower apparent Km values and apparent Vmax values than the unmodified enzyme. So, free PEP behaves as an excellent activator of the reaction at subsaturating substrate concentrations, and as an inhibitor at saturating substrate concentrations. These findings may have important physiological implications on the regulation of the PEP carboxylase in vivo activity and, consequently, of the C4 pathway, since increased reaction rates would be obtained when the concentration of PEP rises, even at limiting Mg2+ concentrations.

Binding Sites↗

PubMed1999

Plausible phosphoenolpyruvate binding site revealed by 2.6 A structure of Mn2+-bound phosphoenolpyruvate carboxylase from Escherichia coli.

We have determined the crystal structure of Mn2+-bound Escherichia coli phosphoenolpyruvate carboxylase (PEPC) using X-ray diffraction at 2.6 A resolution, and specified the location of enzyme-bound Mn2+, which is essential for catalytic activity. The electron density map reveals that Mn2+ is bound to the side chain oxygens of Glu-506 and Asp-543, and located at the top of the alpha/beta barrel in PEPC. The coordination sphere of Mn2+ observed in E. coli PEPC is similar to that of Mn2+ found in the pyruvate kinase structure. The model study of Mn2+-bound PEPC complexed with phosphoenolpyruvate (PEP) reveals that the side chains of Arg-396, Arg-581 and Arg-713 could interact with PEP.

Animals↗

PubMed1998

Studies of the allosteric properties of maize leaf phosphoenolpyruvate carboxylase with the phosphoenolpyruvate analog phosphomycin as activator.

The antibiotic phosphomycin (1,2-epoxypropylphosphonic acid), an analog of phosphoenolpyruvate (PEP), behaved not as an inhibitor, but as an activator, of the enzyme phosphoenolpyruvate carboxylase (PEPC) from maize leaves. Multiple activation studies indicated that the analog binds to the Glc6P-allosteric site producing a more activated enzyme than Glc6P itself. Because of this, we used phosphomycin as a tool to further extend our understanding of the mechanisms of allosteric regulation of C4-PEPC. Initial velocity data from detailed kinetic studies, in which the concentrations of free and Mg-complexed PEP and phosphomycin were controlled, are consistent with: (1) the true activator is free phosphomycin, which competes with free PEP for the Glc6P-allosteric site; and (2) the Mg-phosphomycin complex caused inhibition by binding to the active site in competition with MgPEP. Therefore, although the Glc6P-allosteric site and the active site are able to bind the same ligands, they differ in the form of substrate and activator they bind. This important difference allows the full expression of the potential of activation and prevents inhibition by the activators, including the physiological ones, which are mostly uncomplexed at physiological free Mg2+ concentrations. At fixed low substrate concentrations, the saturation kinetics of the enzyme by phosphomycin showed positive cooperativity at pH 7.3 and 8.3, although at the latter pH, the kinetics of saturation by the substrate was hyperbolic. The cosolute glycerol greatly increased the affinity of the enzyme for phosphomycin and abolished the cooperativity in its binding, but did not eliminate the heterotropic effects of the activator. Therefore, the heterotropic and homotropic effects of the activator are not always coupled to the homotropic effects of the substrate, which argues against the two-state model previously proposed to explain the allosteric properties of maize-leaf PEPC.

Allosteric Regulation↗

PubMed1985

1H and 31P relaxation rate studies of the interaction of phosphoenolpyruvate and its analogues with avian phosphoenolpyruvate carboxykinase.

The interactions of the substrate phosphoenolpyruvate and the substrate analogues (Z)-phosphoenol-alpha-ketobutyrate and (E)-phosphoenol-alpha-ketobutyrate with the enzyme-Mn complex of chicken liver phosphoenolpyruvate carboxykinase have been investigated by 1H and by 31P nuclear relaxation rate studies. Studies of the 1H and the 31P relaxation rates of the ligands in the binary Mn-ligand complexes show that these ligands interact with the metal ion via the phosphate group but not through the carboxylate. An inner sphere coordination complex is formed but the metal-ligand complex is not in the most extended conformation. In the relaxation rate studies of the ligands in the presence of the enzyme, conditions were adjusted so that all of the Mn2+ that was added resided in the ternary enzyme-Mn-ligand complex. The 1H relaxation rates for each of the three ligands were measured at 100 and at 300 MHz. In each case the normalized paramagnetic effects showed that 1/(pT2p) was greater than 1/(pT1p). A frequency dependence of the 1/(pT1p) and 1/(pT2p) values was also measured. The correlation time, tau c, for the Mn-1H interaction was calculated from the frequency dependence of 1/(pT1p) assuming a maximal frequency dependence of tau c and assuming no frequency dependence of tau c and from the T1M/T2M ratios at each frequency. The tau c values for all of the complexes, calculated at 100 MHz, varied from approximately 0.3 to 2.0 ns. These values were used to calculate the Mn-1H distances in each of the ternary complexes. The relaxation rates of 31P were also measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2007

Effect of LiCl on phosphoenolpyruvate carboxylase kinase and the phosphorylation of phosphoenolpyruvate carboxylase in leaf disks and leaves of Sorghum vulgare.

In the present work, the effect of LiCl on phosphoenolpyruvate carboxylase kinase (PEPCase-k), C4 phosphoenolpyruvate carboxylase (PEPCase: EC 4.1.1.31) and its phosphorylation process has been investigated in illuminated leaf disks and leaves of the C4 plant Sorghum vulgare. Although this salt induced severe damages to older leaves, it did not significantly alter the physiological parameters (photosynthesis, transpiration rate, intercellular CO2 concentration) of young leaves. An immunological approach was used to demonstrate that the PEPCase-k protein accumulated rapidly in illuminated leaf tissues, consistent with the increase in its catalytic activity. In vivo, LiCl was shown to strongly enhance the light effect on PEPCase-k protein content, this process being dependent on protein synthesis. In marked contrast, the salt was found to inhibit the PEPCase-k activity in reconstituted assays and to decrease the C4 PEPCase content and phosphorylation state in LiCl treated plants. Short-term (15 min) LiCl treatment increased IP3 levels, PPCK gene expression, and PEPCase-k accumulation. Extending the treatment (1 h) markedly decreased IP3 and PPCK gene expression, while PEPCase-k activity was kept high. The cytosolic protein synthesis inhibitor cycloheximide (CHX), which blocked the light-dependent up-regulation of the kinase in control plants, was found not to be active on this process in preilluminated, LiCl-treated leaves. This suggested that the salt causes the kinase turnover to be altered, presumably by decreasing degradation of the corresponding polypeptide. Taken together, these results establish PEPCase-k and PEPCase phosphorylation as lithium targets in higher plants and that this salt can provide a means to investigate further the organization and functioning of the cascade controlling the activity of both enzymes.

Inositol 1,4,5-Trisphosphate↗

PubMed1996

Phosphoenolpyruvate mutase catalysis of phosphoryl transfer in phosphoenolpyruvate: kinetics and mechanism of phosphorus-carbon bond formation.

Phosphoenolpyruvate phosphomutase (PEP mutase) from Tetrahymena pyriformis catalyzes the rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate (P-pyr). A spectrophotometric P-pyr assay consisting of the coupled actions of P-pyr decarboxylase, phosphonoacetaldehyde hydrolase, and alcohol dehydrogenase was devised to monitor mutase catalysis. The reaction constants determined for PEP mutase catalyzed conversion of PEP to P-pyr at pH 7.5 and 25 degrees C in the presence of Mg(II) are kcat = 5 s(-1), Km = 0.77 +/- 0.05 mM, and Keq = (2-9) x 10(-4). In the PEP forming direction, kcat = 100 s(-1) and Km = 3.5 +/- 0.1 microM. Retention of stereochemistry at phosphorus and strong inhibition displayed by the pyruvyl enolate analog, oxalate, have been cited as two lines of evidence that PEP mutase catalysis proceeds via a phosphoenzyme-pyruvyl enolate intermediate [Seidel, H. M., & Knowles, J. R. (1994) Biochemistry 33, 5641-5646]. In this study, single turnover reactions of oxalyl phosphate with the PEP mutase were carried out to test the formation of the phosphoenzyme intermediate. If formed. the phosphoenzyme-oxalate complex should be sufficiently stable to isolate. Reaction of the mutase with [32P]oxalyl phosphate in the presence of Mg(II)/Mn(II) cofactor failed to produce a detectable level of the [32P]phosphoenzyme-oxalate complex. In contrast, the same reaction carried out with pyruvate phosphate dikinase (PPDK), an enzyme known to catalyze the phosphorylation of its active site histidine with PEP, occurred at a rate of 4 x 10(-4) s(-1) (15% E-P formed) in the presence Mg(II) and at a rate of 3 x 10(-3) s(-1) (60% E-P formed) in the presence of Mn(II). Both oxalyl phosphate (Ki = 180 +/- 10 microM) and oxalate (Ki = 32 +/- 1O microM) were competitive inhibitors of PEP mutase catalysis, but neither displayed slow, tight binding inhibition. These results do not support the intermediacy of a phosphoenzyme-pyruvyl enolate complex in PEP mutase catalysis.

Animals↗

PubMed2003

A conserved 19-amino acid synthetic peptide from the carboxy terminus of phosphoenolpyruvate carboxylase inhibits the in vitro phosphorylation of the enzyme by the calcium-independent phosphoenolpyruvate carboxylase kinase.

Higher plant phosphoenolpyruvate carboxylase (PEPC) is subject to in vivo phosphorylation of a regulatory serine located in the N-terminal domain of the protein. Studies using synthetic peptide substrates and mutated phosphorylation domain photosynthetic PEPC (C4 PEPC) suggested that the interaction of phosphoenolpyruvate carboxylase kinase (PEPCk) with its target was not restricted to this domain. However, no further information was available as to where PEPCk-C4 PEPC interactions take place. In this work, we have studied the possible interaction of the conserved 19-amino acid C-terminal sequence of sorghum (Sorghum vulgare Pers cv Tamaran) C4 PEPC with PEPCk. In reconstituted assays, a C-terminal synthetic peptide containing this sequence (peptide C19) was found to inhibit the phosphorylation reaction by the partially purified Ca2+-independent PEPCk (50% inhibition of initial activity = 230 microm). This effect was highly specific because peptide C19 did not alter C4 PEPC phosphorylation by either a partially purified sorghum leaf Ca2+-dependent protein kinase or the catalytic subunit of mammalian protein kinase A. In addition, the Ca2+-independent PEPCk was partially but significantly retained in affinity chromatography using a peptide C19 agarose column. Because peptide C15 (peptide C19 lacking the last four amino acids, QNTG) also inhibited C4 PEPC phosphorylation, it was concluded that the amino acid sequence downstream from the QNTG motif was responsible for the inhibitory effect. Specific antibodies raised against peptide C19 revealed that native C4 PEPC could be in two different conformational states. The results are discussed in relation with the reported crystal structure of the bacterial (Escherichia coli) and plant (maize [Zea mays]) enzymes.

Amino Acid Sequence↗

PubMed1985

The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides. Mechanism for transfer of the phosphoryl group from phosphoenolpyruvate to fructose.

The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides is a membrane-bound complex of two enzymes, an integral membrane protein EII and a soluble factor SF. In media of high ionic strength the binding constant of SF to the membranes is 55 nM. Phosphorylation of SF, the first step in the reaction sequence, has no influence on the binding. The second step is the transfer of the phosphoryl group from SF to EII. The physical existence of both phosphorylated SF and EII is demonstrated and it is shown that the phosphoryl group is donated to the next species in the reaction sequence. The molecular mass of SF is 110 kDa. EII is almost completely extracted from the membrane as a dimer. The molecular mass of the monomer is 55 kDa. Both SF and EII possess thiols that are essential for catalysis. The thiol on EII is protected against inactivation by N-ethylmaleimide in the phosphorylated state. Kinetic experiments show that the binding site for fructose on EII is induced by phosphorylation of EII ('ping-pong' mechanism). The affinity constants of the phosphotransferase complex for phosphoenolpyruvate and fructose at infinite concentration of the other substrate are 25 microM and 8 microM, respectively. The fructose phosphorylation rate equation is given as a function of the concentration of the two enzymes and the two substrates.

Autoradiography↗