Substrate Phosphoenolpyruvate Research Articles

UDP-N-acetylmuramic Acid (UDP-MurNAc) Is a Potent Inhibitor of MurA (Enolpyruvyl-UDP-GlcNAc Synthase)

Purified recombinant MurA (enolpyruvyl-UDP-GlcNAc synthase) overexpressed in Escherichia coli had significant amounts of UDP-MurNAc (UDP-N-acetylmuramic acid) bound after purification. UDP-MurNAc is the product of MurB, the next enzyme in peptidoglycan biosynthesis. About 25% of MurA was complexed with UDP-MurNAc after five steps during purification that should have removed it. UDP-MurNAc isolated from MurA was identified by mass spectrometry, NMR analysis, and comparison with authentic UDP-MurNAc. Subsequent investigation showed that UDP-MurNAc bound to MurA tightly, with K(d,UDP)(-)(MurNAc) = 0.94 +/- 0.04 microM, as determined by fluorescence titrations using ANS (8-anilino-1-naphthalenesulfonate) as an exogenous fluorophore. UDP-MurNAc binding was competitive with ANS and phosphate, the second product of MurA, and it inhibited MurA. The inhibition patterns were somewhat ambiguous, likely being competitive with the substrate PEP (phosphoenolpyruvate) and either competitive or noncompetitive with respect to the substrate UDP-GlcNAc (UDP-N-acetylglucosamine). These results indicate a possible role for UDP-MurNAc in regulating the biosynthesis of nucleotide precursors of peptidoglycan through feedback inhibition. Previous studies indicated that UDP-MurNAc binding to MurA was not tight enough to be physiologically relevant; however, this was likely an artifact of the assay conditions.

5-Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive to glyphosate

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the penultimate step of the shikimate pathway, and is the target of the broad-spectrum herbicide glyphosate. Kinetic analysis of the cloned EPSPS from Staphylococcus aureus revealed that this enzyme exerts a high tolerance to glyphosate, while maintaining a high affinity for its substrate phosphoenolpyruvate. Enzymatic activity is markedly influenced by monovalent cations such as potassium or ammonium, which is due to an increase in catalytic turnover. However, insensitivity to glyphosate appears to be independent from the presence of cations. Therefore, we propose that the Staphylococcus aureus EPSPS should be classified as a class II EPSPS. This research illustrates a critical mechanism of glyphosate resistance naturally occurring in certain pathogenic bacteria.

Synthesis and evaluation of a mechanism-based inhibitor of KDO8P synthase

The enzyme 3-deoxy- d- manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and d-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate. In attempts to investigate the lack of antibacterial activity of the most potent inhibitor of KDO8P synthase, the amino phosphonophosphate 3 , we have synthesized its hydrolytically stable isosteric phosphonate analogue 4 and tested it as an inhibitor of the enzyme. The synthesis of 4 was accomplished in a one step procedure by employing the direct reductive amination in aqueous media between unprotected sugar phosphonate and glyphosate. The analogue 4 proved to be a competitive inhibitor of KDO8P synthase with respect to both substrates A5P and PEP binding. In vitro antibacterial tests against a series of different Gram-negative organisms establish that both inhibitors ( 3 and 4 ) lack antibacterial activity probably due to their reduced ability to penetrate the bacterial cell membrane.

Characterization and inhibition study of MurA enzyme by capillary electrophoresis

A capillary electrophoresis-based enzyme assay for UDP- N-acetylglucosamine enolpyruvyl transferase (MurA) is described. This method, based on UV detection, provides baseline separation of one of the reaction products, enolpyruvyl-uridine 5′-diphospho- N-acetylglucosamine (EP-UDP-GlcNAc), from substrates phosphoenolpyruvate (PEP) and uridine 5′-diphospho- N-acetylglucosamine (UDP-GlcNAc) within 4 min. The other product, phosphate, is not detectable by UV at 200 nm. Quantitation of individual components, substrates or product, can be accomplished based on the separated peaks. This methodology was used to determine the Michaelis constant, K m, and product formation rate constant, K cat, for MurA. Additionally, the CE method was used to evaluate the inhibition effects on MurA using one specific compound as an example. By following similar procedures, the apparent K m values in the presence of different inhibitor concentrations were determined. The inhibition constant, K i, can be determined from these apparent K m values. In addition, this CE method can be used to study the inhibition mechanism. The principle of this approach is generally applicable to other enzyme studies.

Influence of allosteric effectors on the kinetics and equilibrium binding of phosphoenolpyruvate (PEP) to phosphoenolpyruvate carboxylase (PEPC) from Zea mays

Phosphoenolpyruvate carboxylase (PEPC) the carbon dioxide processing enzyme of C 4 plants, shows the features of an allosteric enzyme. Allosteric activators such as d-glucose-6-phosphate and glycine increase the affinity of PEPC for its substrate PEP at pH 8.0 and pH 7.0. Allosteric inhibitors like l-malate and l-aspartate predominantly decrease the affinity of the carboxylase for PEP at pH 7.0. This was demonstrated by determination of the enzymatic activity and stopped flow (SF) fluorimetry. The binding reaction of PEP to PEPC from Zea mays was measured using the fluorescence probe 2- p-toluidinonaphthalene-6-sulfonate (TNS). The kinetics are described by an allosteric mechanism with a fast reversible bimolecular binding step of PEP to a high affinity (tensed) form of PEPC, which is in equilibrium with its low affinity (relaxed) form. The influence of allosteric effectors on the conformational transition step is demonstrated in support of the description of the kinetics of PEPC by applying a concerted allosteric mechanism as introduced by Monod, Wyman and Changeux. In summary, we present data for the influence of allosteric activators on the kinetics of PEP binding to PEPC and on the concentration dependence of the isomerisation reaction between two allosteric forms of PEPC.

Crystal Structures of KDOP Synthase in Its Binary Complexes with the Substrate Phosphoenolpyruvate and with a Mechanism-Based Inhibitor,

The crystal structures of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli complexed with the substrate phosphoenolpyruvate (PEP) and with a mechanism-based inhibitor (K(d) = 0.4 microM) were determined by molecular replacement using X-ray diffraction data to 2.8 and 2.3 A resolution, respectively. Both the KDOPS.PEP and KDOPS.inhibitor complexes crystallize in the cubic space group I23 with cell constants a = b = c = 117.9 and 117.6 A, respectively, and one subunit per asymmetric unit. The two structures are nearly identical, and superposition of their Calpha atoms indicates an rms difference of 0.41 A. The PEP in the KDOPS.PEP complex is anchored to the enzyme in a conformation that blocks its si face and leaves its re face largely devoid of contacts. This results from KDOPS's selective choice of a PEP conformer in which the phosphate group of PEP is extended toward the si face. Furthermore, the structure reveals that the bridging (P-O-C) oxygen atom and the carboxylate group of PEP are not strongly hydrogen-bonded to the enzyme. The resulting high degree of negative charge on the carboxylate group of PEP would then suggest that the condensation step between PEP and D-arabinose-5-phosphate (A5P) should proceed in a stepwise fashion through the intermediacy of a transient oxocarbenium ion at C2 of PEP. The molecular structural results are discussed in light of the chemically similar but mechanistically distinct reaction that is catalyzed by the enzyme 3-deoxy-D-arabino-2-heptulosonate-7-phosphate synthase and in light of the preferred enzyme-bound states of the substrate A5P.

Evolution of C4 Phosphoenolpyruvate Carboxylase inFlaveria, a Conserved Serine Residue in the Carboxyl-terminal Part of the Enzyme Is a Major Determinant for C4-specific Characteristics

C4 phosphoenolpyruvate carboxylases have evolved from ancestral C3 isoforms during the evolution of angiosperms and gained distinct kinetic and regulatory properties compared with the C3 isozymes. To identify amino acid residues and/or domains responsible for these C4-specific properties the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia (C4) was compared with its orthologue in the closely related C3 plant Flaveria pringlei. Reciprocal enzyme chimera were constructed and the kinetic constants, K(0.5) and k(cat), as well as the Hill coefficient, h, were determined for the substrate phosphoenolpyruvate both in the presence and absence of the activator glucose 6-phosphate. By this approach two regions were identified which determined most of the kinetic differences of the C4 and C3 ppcA phosphoenolpyruvate carboxylases with respect to the substrate PEP. In addition, the experiments suggest that the two regions do not act additively but interact with each other. The region between amino acids 296 and 437 is essential for activation by glucose 6-phosphate. The carboxyl-terminal segment between amino acids 645 and 966 contains a C4 conserved serine or a C3 invariant alanine at position 774 in the respective enzyme isoform. Site-directed mutagenesis shows that this position is a key determinant for the kinetic properties of the two isozymes.

Probing the Stereochemistry of E. coli 3-Deoxy-d-arabino-heptulosonate 7-Phosphate Synthase (Phenylalanine-Sensitive)-Catalyzed Synthesis of KDO 8-P Analogues

The five-carbon phosphorylated monosaccharide analogues, D-arabinose 5-phosphate, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate, were separately condensed with (Z)- and (E)-[3-(2)H]-phosphoenolpyruvate (PEP) in the presence of Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (phe) to give in the case of (Z)-[3-(2)H]-PEP (3S)-[3-(2)H]-3-deoxy-D-manno-octulosonate 8-phosphate, (3S)-[3-(2)H]-3-deoxy-D-altro-octulosonate 8-phosphate, and (3S)-[3-(2)H]-3,5-dideoxy-D-altro-octulosonate 8-phosphate, respectively, whereas incubation with (E)-[3-(2)H]-PEP gives the corresponding (3R)-monosaccharides. These results are in complete agreement with the observed facial selectivity of DAH 7-P synthase for its normal substrates D-erythrose 4-phosphate and PEP and provide direct evidence that DAH 7-P synthase (phe) catalyzes the si face addition of the C3 of PEP to the re face of C1 of the phosphorylated monosaccharides tested. Products formed by DAH 7-P synthase (phe)-catalyzed condensation of (Z)- and (E)-[3-F]-PEP with E 4-P were completely characterized by (1)H and (19)F NMR analysis for the first time. Results of our studies suggest that disappearence of the double bond between C2 and C3 of PEP and formation of a bond between C3 of PEP and C1 of the phosphorylated monosaccharide tested occur in concert during the DAH 7-P synthase-catalyzed condensation reaction.

Structure and Mechanism of 3-Deoxy-d-manno-octulosonate 8-Phosphate Synthase

3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3-deoxy-D-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 A (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (beta/alpha)(8) barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 A apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy.

Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein.

We report here that the E7 oncoprotein encoded by the oncogenic human papillomavirus (HPV) type 16 binds to the glycolytic enzyme type M2 pyruvate kinase (M2-PK). M2-PK occurs in a tetrameric form with a high affinity to its substrate phosphoenolpyruvate and a dimeric form with a low affinity to phosphoenolpyruvate, and the transition between both conformations regulates the glycolytic flux in tumor cells. The glycolytic intermediate fructose 1, 6-bisphosphate induces the reassociation of the dimeric to the tetrameric form of M2-PK. The expression of E7 in an experimental cell line shifts the equilibrium to the dimeric state despite a significant increase in the fructose 1,6-bisphosphate levels. Investigations of HPV-16 E7 mutants and the nononcogenic HPV-11 subtype suggest that the interaction of HPV-16 E7 with M2-PK may be linked to the transforming potential of the viral oncoprotein.

Kinetics and equilibrium binding of phosphoenolpyruvate to phosphoenolpyruvate carboxylase from Zea mays

Phosphoenolpyruvate carboxylase (PEPC) the carbon dioxide processing enzyme of C4 plants shows different affinities for the substrate phosphoenolpyruvate (PEP) at pH 7.0 and pH 8.0. This has been demonstrated by determination of the enzymatic activity, applying fluorescence titrations and fast reaction techniques such as iodine laser temperature jump (ILTJ) and stopped flow (SF). The binding reaction of PEP to PEPC from Zeamays was measured using the fluorescence probe 2-p-toluidinonaphthalene-6-sulfonate (TNS). The kinetics are described by an allosteric mechanism with a fast reversible bimolecular binding reaction of PEP to a high affinity (tensed) form of PEPC which is in equilibrium with its low affinity (relaxed) form. The association and dissociation rate constants k+A and k-A for the fast binding reaction to the high affinity form were determined to be 1.4±0.15×104 M-1 s-1 and 17±6 s-1 at pH 8.0. The corresponding dissociation constants Kd=1.2±0.5 mM for PEP calculated from the kinetic constants, measured by ILTJ and SF, are in good agreement with Kd values achieved in our equilibrium titration experiments or from the data of Michaelis–Menten-type kinetic experiments. PEP preferentially binds to the high affinity binding site of PEPC, shifting the isomerisation equilibrium strongly towards the tensed form, with the consequence that PEPC is activated. Rate constants for the isomerisation process were obtained as kB+(0)=4.95±0.35 s-1 and kB-(0)=1.25±0.1 s-1 at pH 8. Our kinetic data are consistent with the concerted sequential allosteric mechanism introduced by Monod, Wyman and Changeux. In summary, in this study we present, for the first time, data on the kinetics of PEP binding and on the rate of the isomerisation reaction between the two allosteric forms of PEPC.

Cooperativity in Bacillus stearothermophilus pyruvate kinase

The enzyme pyruvate kinase (PK) from the moderate thermophile Bacillus stearothermophilus has been used as a model system with which to investigate the homotropic and heterotropic cooperative interactions of the enzyme. Cooperative ligand binding by the wild-type enzyme was measured using pre-steady-state and steady-state fluorescence spectroscopy, and steady-state kinetics. The results suggest that the cooperative structural changes induced by the substrate phosphoenolpyruvate (PEP) are distinct from those induced by the allosteric activator ribose-5-phosphate (R5P). Furthermore the structural transition induced by the binding of saturating amounts of both PEP and R5P is itself distinct. This conclusion was further substantiated by the production of five mutant proteins in which the R5P- and PEP-induced homotropic cooperative transitions were separated. These results suggest that the cooperativity exhibited by pyruvate kinase from B. stearothermophilus does not conform to a simple two-state model. A putative four-state model is proposed.

First Nonenzymatic Synthesis of Kdo8P through a Mechanism Similar to That Suggested for the Enzyme Kdo8P Synthase

The mechanism of Kdo8P synthase, the enzyme that catalyzes the unusual condensation of d-arabinose 5-phosphate (A5P) with phosphoenolpyruvate (PEP) to form Kdo8P, remains a fascinating subject for bioorganic research. This paper describes the synthesis of two intramolecular models (1 and 2) bearing an enolpyruvate moiety at C-3 of the arabinose fraction. This means that their open-chain aldehyde forms closely mimic the proposed situation, whereby two substrates A5P and PEP evolve into a ternary complex with the synthase. Examination of 1 (in organic solvent) and 2 (in a water solution) under Lewis acid conditions establishes that they both undergo highly stereospecific intramolecular condensation of the enolpyruvate double bond with the carbonyl of sugar. This results in the required Kdo structure possessing the desired stereochemistry. Mechanistic studies suggest that the observed intramolecular condensation process takes place via a stepwise mechanism involving the formation of a transient oxocarbenium ion intermediate. The results obtained, uniquely demonstrate enzyme-like chemistry in the stereospecific synthesis of the Kdo system. Further investigation is certainly warranted, in order to facilitate the construction of other 3-deoxy-2-ulosonoc acids and sialic acids on the basis of the same general model. This is illustrated here in the case of Kdo. Furthermore, the results support the validity of the mechanism suggested for the Kdo8P synthase action, in particular, the possible role of the enzyme in the catalysis of the initial condensation step.

Comparison of the phosphofructokinase and pyruvate kinase activities of Cryptosporidium parvum, Eimeria tenella and Toxoplasma gondii

Oocysts of Cryptosporidium parvum were shown to contain a pyrophosphate-dependent phosphofructokinase (PP i-PFK) similar to those previously described for Eimeria tenella and Toxoplasma gondii. PP i-PFK of C. parvum displayed simple hyperbolic kinetics with respect to its substrate fructose 6-phosphate and was not affected by fructose 2,6-bisphosphate, the major allosteric activator of most ATP-PFKs. Inorganic pyrophosphatase was not detectable in any of the three parasites. T. gondii tachyzoites and C. parvum cysts both contained a pyruvate kinase (PK) specific for ADP rather than PP i AMP . The PK of T. gondii was similar to that of E. tenella in that it displayed strong positive cooperativity with respect to its substrate phosphoenolpyruvate and was heterotropically activated by glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate. PK of C. parvum showed no evidence of allosteric properties. The results suggest that the three coccidia are similar in depending heavily on anaerobic energy production via glycolysis but that the mechanisms for regulating glycolysis are not common to all species.

Essential cysteines in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase from Escherichia coli: analysis by chemical modification and site-directed mutagenesis.

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase (EC 4.1. 2.16) (KDO 8-P synthase) that catalyzes the condensation of D-arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) and inorganic phosphate (Pi) was inactivated by the thiol-modifying reagents 5,5-dithiobis (2-nitrobenzoate) (DTNB) and methyl methanethiosulfonate (MMTS). Reaction of cloned native KDO 8-P synthase with DTNB correlated with modification of two of the four cysteine sulfhydryls per monomer of enzyme and total loss of enzymatic activity which could be partially restored by treatment with dithiothreitol (DTT). Cyanolysis of the DTNB-inactivated enzyme with KCN led to the elimination of 2 equiv of 5-thio-2-nitrobenzoate and partial recovery of activity. The presence of either substrate(s) or product(s) provided no protection against inactivation nor affected the number of cysteines modified, indicating that the cysteines modified are most likely not at the active site of KDO 8-P synthase. Titration of denatured enzyme with DTNB resulted in the modification of all four cysteines. After treatment of native enzyme with MMTS, no cysteines could be titrated with DTNB and no enzymatic activity could be detected. Treatment of the MMTS-inactivated KDO 8-P synthase with DTT resulted in restoration of enzymatic activity and the presence of two DTNB-titratable cysteine residues. Based on these observations and a report that KDO 8-P synthase is inactivated in a time-dependent manner with 3-bromopyruvate and that the substrate PEP protects against this inactivation, all four cysteines (38, 166, 206, and 249) were individually mutated to alanines via a modified PCR methodology. The C206A and C249A mutants were both enzymatically active with K(m) and Vmax values approximately identical to those of wild-type KDO 8-P synthase, and both native mutants reacted with DTNB to modify only one of the three remaining cysteine sulfhydryls per monomer of enzyme. Titration of denatured C206A and C249A mutants resulted in the modification of three cysteines. The C38A and C166A mutants were both for the most part enzymatically inactive. Titration of native C38A and C166A with DTNB resulted in modification of two cysteines while titration of the denatured mutant protein resulted in modification of the three remaining cysteines. Circular dichroism measurements of wild-type KDO 8-P synthase and the four C --> A mutants indicate modest but significant changes in the structure of the mutants. These results indicate that C206 and C249 in native KDO 8-P synthase are readily accessible to the modification reagent DTNB and therefore inactivation may result from structural changes in the DTNB-modified KDO 8-P synthase or blockage of access of substrates to the active site. The C38 and C166 in native KDO 8-P synthase are inaccessible to the modification reagent DTNB, indicating that they are located in the interior of KDO 8-P synthase, and loss of activity in the C38A and C166A mutants suggests their essentiality in the KDO 8-P synthase reaction.

Influence of phosphoenolpyruvate and magnesium ions on the quaternary structure of enzyme I of the phosphotransferase system from gram-positive bacteria.

Solution X-ray scattering patterns of enzyme I of the phosphotransferase system from Staphylococcus carnosus indicate an increase in radius of gyration and molecular mass in the presence of Mg2+ or both Mg2+ and phosphoenolpyruvate, indicating a partial dimerization of enzyme I. Mg2+ ions are essential for both the dimerization and the activation, whereas the substrate phosphoenolpyruvate shifts the monomer--dimer equilibrium to the enzymatically active dimer by decreasing the dissociation rate of the phosphorylated dimer.

An Engineered Change in the L-Malate Sensitivity of a Site-Directed Mutant of Sorghum Phospho enolpyruvate Carboxylase: The Effect of Sequential Mutagenesis and S-Carboxymethylation at Position 8

A recombinant, site-directed mutant form of sorghum phospho enolpyruvate carboxylase (PEPC), in which the phosphorylatable serine residue (Ser-8) was changed to cysteine (S8C), was chemically modified by iodoacetic acid and iodoacetamide for the purpose of testing the effect of introducing a negative charge at position 8. S-Carboxymethylation of the Cys-8 enzyme by iodoacetic acid decreased its sensitivity to L-malate from I 0.5 (50% inhibition) value of 0.12 to 0.35 mM at pH 7.3 when the active-site domain was protected during modification by the substrate phospho enolpyruvate (PEP). In contrast, neither S-carboxymethylation of the wild-type enzyme nor modification of the mutant enzyme by iodoacetamide caused any change in the enzyme′s sensitivity to L-malate. The modified, substrate-protected forms of the Ser-8 and S8C PEPCs had K m (total PEP) and V max values virtually identical to those of the unmodified control enzymes. Similar specific increases in the I 0.5 value of L-malate have been reported previously for in vitro phosphorylated leaf and recombinant Ser-8 PEPCs, the site-directed mutant Asp-8 enzyme, and C 4-leaf PEPC purified from light-adapted sorghum or maize ( in vivo phospho-form). Therefore, these data from different but complementary experimental approaches provide convincing evidence that the effect of phosphorylation of Ser-8 on the L-malate sensitivity of sorghum C 4-PEPC is caused by the introduction of negative charge into this N-termninal regulatory domain.

Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum

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 Km 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.

Characterization of phosphoenolpyruvate carboxykinase fromCorynebacterium glutamicum

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 Km 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.

Purification and characterization of phosphoenolpyruvate carboxykinase, a catabolic CO2-fixing enzyme, from Anaerobiospirillum succiniciproducens

Phosphoenolpyruvate (PEP) carboxykinase (EC 4.1.1.49) from the obligate anaerobe Anaerobiospirillum succiniciproducens was purified 18-fold. The enzyme was monomeric, with an Mr of 57,000 +/- 2,000. The enzyme was oxygen stable, had a pH optimum of 6.7-7.1, and was stable from pH 5.0 to 9.0. The enzyme displayed Michaelis-Menten kinetics for the substrate PEP and the cosubstrates bicarbonate and ADP with a Km of 0.54 mM, 17 mM and 0.42 mM, respectively. The enzyme required Mn(2+) or Co(2+) in addition to Mg(2+) to exhibit maximum activity. p-Chloromercuribenzoate inhibited activity and phosphoenolpyruvate protected the enzyme against inactivation, suggesting that an essential cysteine may be in the active site.