Ca2+-mediated activation of phosphoenolpyruvate carboxykinase occurs via release of Fe2+ from rat liver mitochondria

The addition of calcium chloride to rat liver homogenates resulted in activation of phosphoenolpyruvate carboxykinase by as much as 50%. The enhanced activity was inhibited by quinolinic acid; it was not additive with activation by FeCl2, and stimulation was prevented by 1,10-phenanthroline. Activation by calcium was lost when the particulate fractions of liver were removed, but an activating system could be reconstituted with isolated mitochondria, purified P-enolpyruvate carboxykinase, and purified ferroactivator. Iron-loaded mitochondria were more responsive to calcium than controls. A release of Fe2+ from washed mitochondria could be detected spectrophotometrically when 25-75 nmol of Ca/mg of protein were added to the mitochondrial suspension. If Ca2+ was buffered with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, the threshold of Ca2+ necessary for release of Fe2+ was approximately 10(-7) M, with peak response between 5 X 10(-7) and 10(-6) M. Total Fe2+ detected was normally 20-30 pmol of Fe2+/mg of protein. The synthetic activator of P-enolpyruvate carboxykinase, 3-aminopicolinic acid, as well as other picolinic acid derivatives, is capable of withdrawing Fe2+ associated with the mitochondrial fraction; after incubation with mitochondria, 3-aminopicolinate will activate phosphoenolpyruvate carboxykinase in the absence of exogenous metal.     

Crystallization of the calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli K12

Single crystals of phosphoenolpyruvate carboxykinase from Escherichia coli K12 have been grown in the orthorhombic crystal system. Single crystals developed to a maximum size of 0.25 mm x 0.25 mm x 1.5 mm by the technique of washing and reseeding. The space group is P2(1)2(1)2(1), with a = 77.24 A, b = 89.18 A, c = 93.24 A and Z = 4; there is one enzyme molecule per crystallographic asymmetric unit and the solvent content is estimated to be 59%. The crystals diffract to at least 2.8 A d spacings and decompose in the X-ray beam after approximately two days of exposure.     

Kinetic and structural analysis of Escherichia coli phosphoenolpyruvate carboxykinase mutants

BackgroundPhosphoenolpyruvate carboxykinase (PEPCK) is a metabolic enzyme in the gluconeogenesis pathway, where it catalyzes the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO₂. The substrates for Escherichia coli PEPCK are OAA and MgATP, with Mn²⁺ acting as a cofactor. Analysis of PEPCK structures have revealed amino acid residues involved in substrate/cofactor coordination during catalysis.MethodsKey residues involved in coordinating the different substrates and cofactor bound to E. coli PEPCK were mutated. Purified mutant enzymes were used for kinetic assays. The structure of some mutant enzymes were determined using X-ray crystallography.ResultsMutation of residues D269 and H232, which comprise part of the coordination sphere of Mn²⁺, reduced k_cat by 14-fold, and significantly increased the K_m values for Mn²⁺ and OAA. Mutation of K254 a key residue in the P-loop motif that interacts with MgATP, significantly elevated the K_m value for MgATP and reduced k_cat. R65 and R333 are key residues that interacts with OAA. The R65Q and R333Q mutations significantly increased the K_m value for OAA and reduced k_cat respectively.ConclusionsOur results show that mutation of residues involved in coordinating OAA, MgATP and Mn²⁺ significantly reduce PEPCK activity. K254 plays an important role in phosphoryl transfer, while R333 is involved in both OAA decarboxylation and phosphoryl transfer by E. coli PEPCK.General significanceIn higher organisms including humans, PEPCK helps to regulate blood glucose levels, hence PEPCK is a potential drug target for patients with non-insulin dependent diabetes mellitus.     

Activation of liver cytosol phosphoenolpyruvate carboxykinase by Ca2+ through intracellular redistribution of Mn2+

Calcium has no known direct effect on phosphoenolpyruvate carboxykinase from rat liver cytosol. However, addition of calcium salts to liver postnuclear supernatant led to an increase in assayable enzyme activity in cytosols. This indicates that mitochondria and microsomes present in postnuclear supernatant can participate in observed enzyme activation. The stimulation of phosphoenolpyruvate carboxykinase was prevented by the manganese complexion 1-(2-pyridylazo)-2-naphthol, was not additive with activation by MnCl2 and was inhibited by La3+, Sr2+ and ruthenium red. These data indicate that manganese and mitochondrial or microsomal calcium carriers participate in the mechanism of indirect calcium effect. Measuring of manganese content in cytosols directly, by atomic absorption spectrometry, has provided evidence that there is a pool of manganese associated with mitochondrial and microsomal fraction of rat liver that can be mobilized to the cytosol by calcium ions. The direct addition of this pool of manganese to the cytosol caused the stimulation of phosphoenolpyruvate carboxykinase activity to the same levels as did calcium ions in the postnuclear supernatant. It is postulated that calcium can effect enzyme activity indirectly by releasing manganese from specific cellular compartments into the cytosol.     

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.     

Temperature-sensitive mutation affecting synthesis of phosphoenolpyruvate carboxykinase in Escherichia coli.

A mutation has been characterized in Escherichia coli which results in temperature-sensitive expression of phosphoenolpyruvate carboxykinase activity and antigen. The enzyme produced by the mutant strain at a permissive temperature or by cells treated with chloramphenicol at nonpermissive temperatures had normal activity and stability in extracts. Since phosphoenolpyruvate carboxykinase had a monomeric structure, the mutation probably affects the synthesis, rather than the structure or assembly, of the enzyme.     

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.     

A mutant phosphoenolpyruvate carboxykinase in Escherichia coli conferring oxaloacetate decarboxylase activity.

The phosphoenolpyruvate carboxykinase in Escherichia coli (encoded by pck) catalyzes the conversion from oxaloacetate (OAA) to phosphoenolpyruvate under gluconeogenic conditions. We report here the characterization of two mutant alleles, pck-51 and pck-53, both of which are point mutations leading to single amino acid changes (D to N at position 268 and G to S at position 284, respectively). Pck51 is an altered-activity mutant that catalyzes the conversion from OAA to pyruvate (OAA decarboxylase activity). This new activity was not detected from the wild-type Pck, and it complements the pck null mutation only in a pps+ background. Pck53 is a reduced-activity mutant that complements the pck null mutation in a strain-dependent fashion.     

The phosphoryl-transfer mechanism of Escherichia coli phosphoenolpyruvate carboxykinase from the use of AlF(3).

The mechanism of reversible transfer of the gamma-phosphate group of ATP by Escherichia coli phosphoenolpyruvate carboxykinase (PCK) on to its substrate is of great interest. It is known that metallofluorides are accurate analogs of the transition state in the context of kinase mechanisms. Therefore, two complexes of PCK, one with AlF(3), Mg(2+) and ADP (complex I), the other with AlF(3), Mg(2+), ADP and pyruvate (complex II) were crystallized. The X-ray crystal structures of these two complexes were determined at 2.0 A resolution. The Al atom has trigonal bipyramidal geometry that mimics the transition state of phosphoryl transfer. The Al atom is at a distance of 2.8 A and 2.9 A from an oxygen atom of the beta-phosphoryl group of ADP in complex I and II, respectively. A water molecule in complex I and an oxygen atom of the pyruvate in complex II are located along the axis of the trigonal bipyramid on the side opposite to the beta-phosphoryl oxygen with respect to the equatorial plane, suggesting that the complexes are close mimics of the transition state. Along with the presence of positively charged species around the AlF(3) moiety, these results indicate that phosphoryl transfer occurs via a direct displacement mechanism with associative qualities.     

Ligand interactions and protein conformational changes of phosphopyridoxyl-labeled Escherichia coli phosphoenolpyruvate carboxykinase determined by fluorescence spectroscopy.

Escherichia coli phosphoenolpyruvate (PEP) carboxykinase catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-phosphoryl group of ATP to yield PEP, ADP, and CO2. The interaction of the enzyme with the substrates originates important domain movements in the protein. In this work, the interaction of several substrates and ligands with E. coli PEP carboxykinase has been studied in the phosphopyridoxyl (P-pyridoxyl)-enzyme adduct. The derivatized enzyme retained the substrate-binding characteristics of the native protein, allowing the determination of several protein-ligand dissociation constants, as well as the role of Mg2+ and Mn2+ in substrate binding. The binding affinity of PEP to the enzyme-Mn2+ complex was -8.9 kcal.mol-1, which is 3.2 kcal.mol-1 more favorable than in the complex with Mg2+. For the substrate nucleotide-metal complexes, similar binding affinities (-6.0 to -6.2 kcal.mol-1) were found for either metal ion. The fluorescence decay of the P-pyridoxyl group fitted to two lifetimes of 5.15 ns (34%) and 1.2 ns. These lifetimes were markedly altered in the derivatized enzyme-PEP-Mn complexes, and smaller changes were obtained in the presence of other substrates. Molecular models of the P-pyridoxyl-E. coli PEP carboxykinase showed different degrees of solvent-exposed surfaces for the P-pyridoxyl group in the open (substrate-free) and closed (substrate-bound) forms, which are consistent with acrylamide quenching experiments, and suggest that the fluorescence changes reflect the domain movements of the protein in solution.     

On the monomeric structure and proposed regulatory properties of phosphoenolpyruvate carboxykinase of Escherichia coli.

Phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating)) (EC 4.1.1.49) has been purified to homogeneity from Escherichia coli. The enzyme shows the same molecular weight (ca. 65000) either by sedimentation equilibrium under nondenaturing conditions or by polyacrylamide gel electrophoresis in the presence of detergent, indicating that the enzyme has a monomeric structure. We have confirmed the previous observation that NADH is an inhibitor of this enzyme, but we have failed to detect the previously reported appearance of homotropic cooperativity with respect to substrate binding the presence of this inhibitor. Lack of such homotropic interactions is in harmony with our conclusion that the enzymes is a monomer. Replacement of Mg2+ by Mn2+ in the assay medium lowers the Km for phosphoenolpyruvate by an order of magnitude, but does not affect the characteristics of inhibition by NADH.     

Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation. In contrast, in the phosphoenolpyruvate carboxylase mutant E. coli strain K-12 ppc::kan, PEPCK overexpression increased succinate production 6.5-fold.     

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.     

L-threonine dehydrogenase from Escherichia coli K-12: thiol-dependent activation by Mn2+

Addition of 1 mM Mn2+ to all solutions in the final chromatographic step used to purify L-threonine dehydrogenase (L-threonine:NAD+ oxidoreductase, EC 1.1.1.103) from extracts of Escherichia coli K-12 routinely provides 30-40 mg of pure enzyme per 100 g wet weight of cells with specific activity = 20-30 units/mg. Enzyme dialyzed exhaustively against buffers containing Chelex-100 resin has a specific activity = 8 units/mg and contains 0.003 or 0.02 mol of Mn2+/mol of enzyme as determined by radiolabeling studies with 54Mn2+ or by atomic absorption spectroscopy, respectively. Dehydrogenase activity is completely abolished by low concentrations of either Hg2+ or Ag+; of a large spectrum of other metal ions tested, only Mn2+ and Cd2+ have an activating effect. Activation of threonine dehydrogenase by Mn2+ is thiol-dependent and is saturable with an activation Kd = 9.0 microM and a Vmax = 105 units/mg. Stoichiometry of Mn2+ binding was found to be 0.86 mol of Mn2+/mol of enzyme subunit with a dissociation constant (Kd) = 8.5 microM. Mn2+ appears to interact directly with threonine dehydrogenase; gel filtration studies with the dehydrogenase plus 54Mn2+ in the presence of either NAD+, NADH, L-threonine, or combinations thereof show that only Mn2+ coelutes with the enzyme whereas all other ligands elute in the salt front and the stoichiometry of the dehydrogenase-Mn2+ interaction is not affected in any instance. A theoretical curve fit to data for the pH-activity profile of Mn2+-saturated enzyme has a pKa = 7.95 for one proton ionization. The data establish L-threonine dehydrogenase of E. coli to be a metal ion activated enzyme.