Purification and molecular characteristics of mitochondrial phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver

Phosphoenolpyruvate carboxykinase from bullfrog liver mitochondria has been purified to electrophoretical and immunological homogeneity by an improved method using hydrophobic chromatography on Sepharose-hexane-GMP and affinity chromatography on phosphocellulose. The molecular weight was determined to be 70,000 by SDS-gel electrophoresis, 65,000 by Sephadex G-100 gel filtration and 72,000 by glycerol gradient centrifugation. The isoelectric point was determined to be 6.2, differing from that of the cytosol enzyme. The rabbit IgG fraction against the mitochondrial PEP carboxykinase precipitated not only the mitochondrial but also the cytosol enzyme. The dissociation constant of the nucleotide-enzyme complex was determined to be 3 microM for GTP, 8.5 microM for GDP, and 171 microM for GMP. The affinity of GTP for the enzyme was reduced in the presence of phosphoenolpyruvate or Mn2+, whereas that of GDP was not changed. GMP inhibited the enzyme competitively with GDP for the phosphoenolpyruvate carboxylation and competitively with GTP for the exchange reaction between [14C]HCO3- and oxaloacetate. The purified enzyme was found to have a cysteine residue which reacted with iodoacetamide to form inactive enzyme. Guanine nucleotides or IDP and Mn2+ at a lower concentration prevented the inactivation by iodoacetamide of the enzyme in a competitive manner. Binding of guanine nucleotide to the enzyme and the relation of the sulfhydryl group to the nucleotide binding are discussed.     

Phosphoenolpyruvate carboxykinase in mouse pancreatic islets. ATP-induced changes in sensitivity to Mn2+ activation

The presence of high phosphoenolpyruvate carboxykinase (EC 4.1.1.32) activity in mouse islet cytosol has been demonstrated. The enzyme was activated by Mn2+ with a Ka of 100 X 10(-6) mol/l. The mean total activity of the Mn2+-stimulated phosphoenolpyruvate carboxykinase in islet cytosol estimated at 22 degrees C with saturating concentrations of the substrates oxaloacetate and ITP was 146 pmol/min per micrograms DNA. Km was calculated to be 6 X 10(-6) mol/l for oxaloacetate and 140 X 10(-6) mol/l for ITP. The islet phosphoenolpyruvate carboxykinase activity was not increased after starvation of the animals for 48 h. Preincubation of the cytosol at 4 degrees C with Fe2+, quinolinate, ATP, Pi, glucose 6-phosphate, fructose 1,6-bisphosphate, NAD+, NADH, oxaloacetate, ITP, cyclic AMP and Ca2+ had no effect on the enzyme activity. However, preincubation of the cytosol at 37 degrees C with ATP-Mg inhibited the Mn2+-stimulated phosphoenolpyruvate carboxykinase activity progressively with time and in a concentration-dependent manner. A similar but weaker inhibitory effect was observed with p[NH]ppA, whereas p[CH2]ppA, ADP, AMP, adenosine and Pi had no effect. It is tentatively suggested that ATP and p[NH]ppA either by adenylation or otherwise affect the interaction between islet phosphoenolpyruvate carboxykinase and the recently discovered Mr = 29000 protein modulator of the enzyme in such a way - perhaps by causing a dissociation between them - that phosphoenolpyruvate carboxykinase loses its sensitivity to Mn2+ activation.     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.     

Purification and properties of mitochondrial phosphoenolpyruvate carboxykinase from liver of Squalus acanthias

Liver from Squalus acanthias (spiny dogfish), a representative elasmobranch, contains approximately 1.4 units (mumol/min) of phosphoenolpyruvate carboxykinase activity per gram and approximately 90% of the total units of activity are localized in the mitochondria. The mitochondrial phosphoenolpyruvate carboxykinase was isolated and characterized. The purified enzyme has properties generally similar to those found in mammalian and avian species. The enzyme has a molecular weight of approximately 70,000 and exists in a functional state as a monomer. The isolated enzyme displays a dual cation requirement (e.g., 6 mM Mg2+ and 10 microM Mn2+) for maximal activity; very little activity is observed when Mg2+ is present alone, and the maximal activity attained with Mn2+ alone (millimolar concentrations required) is significantly less than that observed under optimal conditions with both cations present. When assayed in the direction of oxalacetate formation there is a lag in product formation with time; the lag can be eliminated by the presence of 50 microM GTP (product). The Km for substrates is not affected by Mn2+ concentration, suggesting that the role of Mn2+ may not be related to substrate binding. The apparent Km for phosphoenolpyruvate (approximately 1 mM) is substantially higher than that reported for phosphoenolpyruvate carboxykinase from other species. The activity of phosphoenolpyruvate carboxykinase is increased 70% by physiological concentrations of urea. Maximal velocity of the reaction in the direction of oxalacetate formation is approximately half that of the reverse reaction.     

Effects of Mn2+ on the exchange reaction of phosphoenolpyruvate carboxykinase in the presence of high concentrations of Mg2+

The mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32), purified from chick embryo liver, was synergistically activated by a combination of Mn2+ and Mg2+ in the oxaloacetate ---- H14CO-3 exchange reaction. Increases in the Mg2+ concentration caused decreases in the K0.5 value of Mn2+ in line with the earlier finding that the enzyme was markedly activated by low Mn2+ (microM) plus high Mg2+ (mM). In the presence of 2.5 mM Mg2+, increases in the Mn2+ level first enhanced the activity of phosphoenolpyruvate carboxykinase, and then suppressed it to the maximal velocity shown in the presence of Mn2+ alone. Kinetic studies showed that high Mn2+ inhibited the activity of Mg2+ noncompetitively, and those of GTP and oxaloacetate uncompetitively. The inhibition constant for oxaloacetate (K'i = 550 microM) was lower than that of Mg2+ (Ki = K'i = 860 microM) or GTP (K'i = 1.6 mM), and was nearly equal to the apparent half-maximal inhibition concentration of Mn2+. These results suggested that Mn2+ can play two roles, of activating and suppressing phosphoenolpyruvate carboxykinase activity in the presence of high Mg2+.     

Effects of phosphate anions and some divalent metal cations on phosphoenolpyruvate carboxykinase. Comparison of liver and kidney enzyme

The effect of calcium and phosphate anions on rat kidney cytosol phosphoenolpyruvate carboxykinase activity was evaluated using enzyme preparations obtained by two purification procedures. The enzyme activity was not significantly affected by calcium ions at physiological concentration. Phosphate inhibited the enzyme in the presence of Fe2+; the inhibition was overcome by Mn2+. Kidney and liver phosphoenolpyruvate carboxykinases show some qualitative differences in their response to Fe2+ and phosphate.     

Effect of triamcinolone on renal and hepatic phosphoenolpyruvate carboxykinase in the newborn rat. Changes in the rate of synthesis of the enzyme and in the activity of its translatable messenger RNA.

The effects of triamcinolone on renal and hepatic phosphoenolpyruvate carboxykinase activity in the developing rat were investigated. The hormone induced increases in pre-existing enzyme activity of both tissues in fetal and neonatal rats, yet did not cause the primary appearance of phosphoenolpyruvate carboxykinase activity in utero. Neonatal hepatic phosphoenolpyruvate carboxykinase activity was increased 2--3 fold by triamcinolone form the 3rd to the 15th postnatal day. This was shown to be additive to the effect of Bt2cAMP on enzyme activity. The increases in phosphoenolpyruvate carboxykinase activity were demonstrated to be due to increased synthesis of the enzyme, which was accompanied by a proportionate increase in the amount of functional phosphoenolpyruvate carboxykinase mRNA, as measured by the polyribosomal and poly(A)-containing RNA directed cell-free synthesis of the enzyme. The demonstration of a triamcinolone effect on kidney and liver phosphoenolpyruvate carboxykinase activity in fetal and neonatal rats provides support for a possible role of glucocorticoids in the regulation of phosphoenolpyruvate carboxykinase activity during development.     

An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.     

Kinetic studies with cytosol and mitochondrial phosphoenolpyruvate carboxykinases

1. Measurements of Michaelis constants for oxaloacetate in the reaction catalysed by liver phosphoenolpyruvate carboxykinase give values much lower than previously reported. With Mg(2+) as bivalent cation, the Michaelis constant was approx. 2.5x10(-5)m whether the enzyme used was the mitochondrial phosphoenolpyruvate carboxykinase purified from sheep liver or chicken liver or the cytosol enzyme purified from rat liver or sheep liver. 2. When Mn(2+) replaced Mg(2+) in the reaction a lower Michaelis constant of 9x10(-6)m was found, but only with the mitochondrial enzymes. 3. With all enzymes malate at high concentration was a competitive inhibitor with respect to oxaloacetate when Mn(2+) was the added cation. With Mg(2+) the inhibition by malate was competitive with the mitochondrial enzymes and non-competitive with the cytosol enzymes.     

Affinity labeling of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with the 2',3'-dialdehyde derivative of ATP

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is completely inactivated by the 2',3'-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 +/- 17 microM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 degrees C is 0.200 +/- 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.     

A histidine residue at the active site of avian liver phosphoenolpyruvate carboxykinase

The histidine-selective reagents diethylpyrocarbonate (DEPC) and dimethylpyrocarbonate were used to study active site residues of phosphoenolpyruvate carboxykinase. Both reagents show pseudo first-order inhibition of enzyme activity at 22 +/- 1 degree C with calculated second-order rate constants of 2.8 and 4.6 M-1 s-1, respectively. The inhibition appears partially reversible. Substrates affect the rate of inhibition: KHCO3 enhances the rate, Mn2+ has little effect, and phosphoenolpyruvate decreases the rate. The best protection is obtained by IDP or IDP and Mn2+. The kinetic studies show that modification of histidine is specific and leads to loss of enzymatic activity. Two histidines per enzyme are modified by DEPC, as measured by an absorption change at 240 nm, in the absence of substrate, leading to loss in activity. One histidine per molecule is modified in the presence of KHCO3, giving inactivation. Cysteine and lysine residues are not affected. A study of the inhibition rate constant as a function of pH gives a pKa of 6.7. Enzyme modified by DEPC in the absence of substrate (1% remaining activity) shows no binding of ITP or of phosphoenolpyruvate to the enzyme.Mn2+ complex as studied by proton relaxation rates. When enzyme is modified in the presence of KHCO3 (44% remaining activity), ITP and KHCO3 bind to the enzyme.Mn2+ complex similarly to the binding to native enzyme. Phosphoenolpyruvate binding to modified enzyme.Mn results in an enhancement of proton relaxation rates rather than the decrease observed with native enzyme.Mn. The CD spectra of histidine-modified enzyme show a decrease in alpha-helical and random structure with an increase in anti-parallel beta-sheet structure compared to native enzyme. These results show that avian phosphoenolpyruvate carboxykinase has 2 histidine residues which are reactive with DEPC and dimethylpyrocarbonate, and one of the 15 histidine residues in the protein is at or near the phosphoenolpyruvate binding site and is involved in catalysis.     

Identification of a phosphorylated form of phosphoenolpyruvate carboxykinase from the yeast Saccharomyces cerevisiae

A phosphoprotein of 65 kDa, as determined by SDS-gel electrophoresis, has been isolated from yeast crude extracts. This phospho form copurifies with phosphoenolpyruvate carboxykinase in the enzyme purification procedure worked out in our laboratory (Tortora, P., Hanozet, G.M. and Guerritore, A. (1985) Anal. Biochem. 144, 179-185). Moreover, both proteins bind strongly to 5'AMP-Sepharose 4B in the presence of Mn2+, whereas a substantially lower binding occurs if Mn2+ is replaced by Mg2+. This binding pattern is consistent with the well-known Mn2+-dependence of yeast phosphoenolpyruvate carboxykinase. These data suggest that the 65-kDa protein might be a phosphorylation product of the native enzyme. Furthermore, although the phospho form is not immunoprecipitated by anti-phosphoenolpyruvate carboxykinase antibodies, addition of Protein A-Sepharose CL-4B to crude extracts preincubated with the antibodies results in the binding to the resin of the phospho form, thus providing immunological evidence for its identification as a modified form of native enzyme. The same 65-kDa phosphoprotein is detectable in extracts from cells grown in the presence of [32P]Pi, as well as in cell extracts incubated with [gamma-32P]ATP. Moreover, digestion of the phosphoprotein with BrCN or with Staphylococcus aureus V8 proteinase, yields two and three fragments, respectively, which appear parallel to digestion products of phosphoenolpyruvate carboxykinase, again supporting the proposed identification. Finally, analysis of the phosphorylated amino acids in the 65-kDa protein shows that phosphoserine is the only labelled phosphoamino acid.     

Purification of phosphoenolpyruvate carboxykinase (GTP) by affinity chromatography on agarose-hydrazide-GTP

The cytosolic form of phosphoenolpyruvate carboxykinase (GTP; EC 4.1.1.32) from rat liver was purified by a procedure involving affinity chromatography on agarose-hydrazide-GTP. Phosphoenolpyruvate carboxykinase is retained quantitatively by the affinity medium in the presence of manganese and can be specifically eluted by a pulse of GTP. On the contrary, no binding to agarose-hydrazide-GTP occurs in the absence of manganese. This suggests that the affinity of the enzyme for GTP is enhanced by prior interaction with manganese. A combination of several conventional purification steps followed by affinity chromatography provides pure phosphoenolpyruvate carboxykinase in good yields. The final specific activity is 19 U/mg protein. The enzyme migrates as a single polypeptide of molecular weight 70,600 during electrophoresis on sodium dodecyl sulfate polyacrylamide gels.     

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.     

Comparison of Phosphoenolpyruvate-Carboxykinase from Autotrophically and Heterotrophically Grown Euglena and Its Role during Dark Anaerobiosis

Euglena gracilis (1224-5/9) contains phosphoenolpyruvate carboxykinase when grown autotrophic with CO₂ in the light. Its yield is higher when an additional carbon source like glucose has been added. The enzyme is lacking in cells provided with CO₂ alone and kept in the dark, whereas highest yields result if both glucose and CO₂ are provided together in the dark. The enzyme was purified by ammonium sulfate precipitation, gel filtration on Sephacryl S-300 and affinity chromatography on GMP-Sepharose. The latter step was most effective to protect the enzyme from inactivation. Its homogeneity was tested electrophoretically and immunologically. Enzymes from autotrophic and heterotrophically grown cells have identical pH optima and similar isoelectric points. The molecular weight was different: 761,000 for the enzyme from autotrophic and 550,000 for that from heterotrophic cells as determined by gel filtration. The subunit molecular weight of both enzymes is nearly the same. The kinetic data of the enzymes are slightly different. Glycolytic and tricarboxylic acid cycle intermediates are of limited influence on enzyme activity and inhibitory in unphysiological high concentrations. From Ouchterlony double immunodiffusion and enzyme-linked immunosorbent assay, it is evident that the enzyme is localized in the cytosol. With the latter quantification test the phosphoenolpyruvate carboxykinase protein content was found 10 times higher in heterotrophically grown cells than when cultivated under autotrophic conditions.     

Synthesis of oxalyl phosphate and processing of the acyl phosphate by phosphoenolpyruvate-dependent enzymes

The mixed anhydride of oxalic and phosphoric acids, oxalyl phosphate, has been prepared by reaction of oxalyl chloride and inorganic phosphate in aqueous solution. The product was purified by anion exchange chromatography and characterized by 31P and 13C NMR. This acyl phosphate has a half-life of 51 h at pH 5.0 and 4 degrees C. Oxalyl phosphate, an analogue of phosphoenolpyruvate, is a slow substrate for pyruvate kinase, undergoing an enzyme-dependent phosphotransfer reaction to produce ATP from ADP. Oxalyl phosphate substitutes for phosphoenolpyruvate in the reaction catalyzed by pyruvate, phosphate dikinase. The acyl phosphate reacts with the free enzyme to give the phosphorylated form of the enzyme. Removal of the potent product inhibitor, oxalate, from the reaction mixtures by gel filtration chromatography permitted further reaction of the phosphorylated enzyme with pyrophosphate and AMP to give ATP and Pi in a single turnover assay. Oxalyl phosphate also served as a phospho group donor in a partial reaction catalyzed by phosphoenolpyruvate carboxykinase wherein GDP is phosphorylated at the expense of oxalyl phosphate.     

Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana

The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.     

3-Mercaptopicolinate. A reversible active site inhibitor of avian liver phosphoenolpyruvate carboxykinase

The inhibition of chicken liver phosphoenolpyruvate carboxykinase by 3-mercaptopicolinic acid (3-MP) has been investigated. Kinetic studies show 3-MP to be a noncompetitive inhibitor relative to all substrates and to the activator, Mn2+. EPR studies demonstrate that Mn2+ binding to the enzyme is unaffected by 3-MP. Proton relaxation rate studies demonstrate that 3-MP binds to the binary E X Mn complex with a KD of 0.5 X 10(-6) M and gives a ternary enhancement of 8.0. Additional proton relaxation rate studies detected formation of the quaternary complexes E X Mn X IDP X 3-MP, E X Mn X ITP X 3-MP, and E X Mn X CO2 X 3-MP. High resolution 1H nuclear relaxation rate studies suggest that 3-MP binds in close proximity to the activator cation, Mn2+, but not in the first coordination sphere. Active site models suggest that the 3-MP-binding site may partially overlap the phosphoenolpyruvate-binding site. The NMR studies, which detected formation of the quaternary E X Mn X 3-MP X phosphoenolpyruvate complex, also demonstrated that the binding of one of these ligands affects the interactions of the other ligand with E X Mn. Calorimetric studies of the E X Mn complex demonstrated that 3-MP causes an increase in the transition temperature midpoint without an increase in enthalpy. These results indicate that 3-MP causes a conformational change in the enzyme but does not increase the thermostability of the ternary complex. The experiments reported herein suggest that inhibition by 3-MP is due to specific and reversible binding within the active site of phosphoenolpyruvate carboxykinase.     

Lysine 213 and histidine 233 participate in Mn(II) binding and catalysis in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO2 and plays a key role in gluconeogenesis. This enzyme also has oxaloacetate decarboxylase and pyruvate kinase-like activities. Mutations of PEP carboxykinase have been constructed where the residues Lys213 and His233, two residues of the putative Mn2+ binding site of the enzyme, were altered. Replacement of these residues by Arg and by Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn2+ binding affinity. Lower PEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against urea denaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in V(max) for the primary reaction relative to that for the wild-type enzyme. V(max) values for the oxaloacetate decarboxylase and pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in the mutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn2+ and PEP. The results are consistent with Lys213 and His233 being at the Mn2+ binding site of S. cerevisiae PEP carboxykinase and the Mn2+ affecting the PEP interaction. The different effects of mutations in V(max) for the main reaction and the secondary activities suggest different rate-limiting steps for these reactions.     

Arginine residues at the active site of avian liver phosphoenolpyruvate carboxykinase

The presence of arginine at the active site of avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification followed by a characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione all irreversibly inhibit the enzyme with second-order rate constants of 3.42 M-1 min-1, 3.13 M-1 min-1 and 0.313 M-1 min-1, respectively. The substrates phosphoenolpyruvate, IDP, and the activator Mn2+ offer little to modest protection from inhibition. Either CO2 or CO2 in the presence of any of the other substrates elicited potent protection against modification. Protection by CO2 against modification by phenylglyoxal or 1,2-cyclohexanedione gave a biphasic pattern. Rapid loss in activity to 40-60% occurred, followed by a very slow loss. Kinetics of inhibition suggest that the modification of arginine is specific and leads to loss of enzymatic activity. Substrate protection studies indicate an arginine residue(s) at the CO2 site of phosphoenolpyruvate carboxykinase. Apparently no arginine residues are at the binding site of the phosphate-containing substrates. Partially inactive (40-60% activity) enzyme, formed in the presence of CO2, has a slight change of its kinetic constants, and no alteration of its binding parameters or secondary structure as demonstrated by kinetic, proton relaxation rate, and circular dichroism studies. Labeling of enzyme with [(7-)14C]phenylglyoxal in the presence of CO2 (40-60% activity) showed 2 mol of phenylglyoxal/enzyme or 1 arginine or cysteine residue modified. Labeling of phosphoenolpyruvate carboxykinase in the absence of CO2 yielded 6 mol of label/enzyme. Labeling results indicate that avian phosphoenolpyruvate carboxykinase has 2 or 3 reactive arginine residues out of a total of 52 and only 1 or 2 are located at the active site and are involved in CO2 binding and activation.