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PubMed1976

Liver cell heterogeneity. The distribution of pyruvate kinase and phosphoenolpyruvate carboxykinase (GTP) in the liver lobule of fed and starved rats.

Pyruvate kinase and phosphoenolpyruvate carboxykinase activities were determined in microdissected freeze-dried liver cells from the periportal and pericentral area of the liver lobule. Pyruvate kinase activity was measured by a microfluorimetric procedure adapted to 20-200 ng tissue dry weight. In livers from fed rats, its activity was twice as high in the central zone as in the periportal cells; starvation reduced this gradient by decreasing central activities. Phosphoenolpyruvate carboxykinase activity was measured by a microradiochemical technique in 100-300 ng tissue dry weight. In livers from fed rats, this enzyme was nearly 3 times more active in the periportal cells than in the central area. Starvation increased this enzyme in both zones with a more pronounced change in the central cells. The results indicate a heterogeneous distribution of enzymes of carbohydrate metabolism in the liver lobule. Gluconegenesis seems to be localized preferentially in periportal hepatocytes, whereas the glycolytic enzyme was found to be more active in cells surrounding the pericentral liver cells.

Animals↗
PubMed1993

Stereochemistry of metal ion coordination to the terminal thiophosphoryl group of adenosine 5'-O-(3-thiotriphosphate) at the active site of pyruvate kinase.

Epimers of [gamma-17O]adenosine 5'-O-(3-thiotriphosphate) ([gamma-17O]ATP gamma S) have been used to determine the stereochemistry of Mn2+ coordination to the terminal thiophosphoryl group in complexes of pyruvate kinase, oxalate, ATP gamma S, and Mg2+, Zn2+, Co2+, or Cd2+. The complex of pyruvate kinase with oxalate and ATP binds 2 equiv of divalent cation per active site. The terminal phosphoryl group of ATP in this enzymic complex becomes a chiral center as a result of coordination to both divalent metal ions. Electron paramagnetic resonance (EPR) data for complexes of pyruvate kinase with Rp- or Sp-[gamma-17O]-ATP gamma S, [17O]oxalate, and mixtures of Mn2+ with Mg2+, Zn2+, or Co2+ show that Mn2+ binds selectively at the site defined by coordination to oxalate and the pro-R oxygen of the thiophosphoryl group of ATP gamma S. In mixtures containing Mn2+ and Cd2+ with Tl+ as the monovalent cation, two hybrid complexes form, enzyme-oxalate-MnII-ATP gamma S-CdII and enzyme-oxalate-CdII-ATP gamma S-MnII, as in the analogous complexes with ATP and K+ or Tl+ (Buchbinder, J. L., & Reed, G. H. (1990) Biochemistry 29, 1799-1806). In the enzyme-oxalate-MnII-ATP gamma S-CdII species, Mn2+ binds exclusively to the pro-R oxygen of the thiophosphoryl group. In the enzyme-oxalate-CdII-ATP gamma S-MnII species, Mn2+ binds to the pro-R oxygen (60%) and to the pro-S oxygen (40%).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1997

Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate.

We have characterized the gene YOR347c of Saccharomyces cerevisiae and shown that it encodes a second functional pyruvate kinase isoenzyme, Pyk2p. Overexpression of the YOR347c/PYK2 gene on a multicopy vector restored growth on glucose of a yeast pyruvate kinase 1 (pyk1) mutant strain and could completely substitute for the PYK1-encoded enzymatic activity. PYK2 gene expression is subject to glucose repression. A pyk2 deletion mutant had no obvious growth phenotypes under various conditions, but the growth defects of a pyk1 pyk2 double-deletion strain were even more pronounced than those of a pyk1 single-mutation strain. Pyk2p is active without fructose-1,6-bisphosphate. However, overexpression of PYK2 during growth on ethanol did not cause any of the deleterious effects expected from a futile cycling between pyruvate and phosphoenolpyruvate. The results indicate that the PYK2-encoded pyruvate kinase may be used under conditions of very low glycolytic flux.

Allosteric Regulation↗
PubMed1986

The regulatory properties of yeast pyruvate kinase. Effect of fructose 1,6-bisphosphate.

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 at 25 degrees C as a function of the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ and the concentration of the effector fructose 1,6-bisphosphate. The enzyme was activated by 100 mM-K+ and 32 mM-NH4+ throughout. It was found that an increase in the fructose bisphosphate concentration from 24 microM to 1.2 mM brings about a transition from a sigmoidal to a non-inflected form in the relationships v = f([phosphoenolpyruvate]) and v = f([Mg2+]) together with a large increase in the affinity of these substrates for the enzyme. The binding behaviour of ADP is barely affected by the same change in effector concentration. By contrast, increase in fructose bisphosphate concentration below 24 microM increases the affinity of the enzyme for all its substrates and the sigmoidicity of the corresponding velocity-substrate-concentration relationships. As a result of this change in behaviour it has been found impossible to represent all the data by the exponential model for a regulatory enzyme, and it is suggested (supported by comparisons with previous work) that the failure may reflect a secondary action of the effector upon the enzyme.

Adenosine Diphosphate↗
PubMed1985

Changes in phosphofructokinase and pyruvate kinase in rat brain following thyroidectomy.

Changes in the profile of phosphofructokinase (PFK) and Pyruvate kinase (PK) from rat brain regions were studied following thyroidectomy. A regional variation of thyroidectomy on brain was observed; cerebral hemispheres and cerebellum showed decreased activity of phosphofructokinase and pyruvate kinase. The activity in the brain stem did not change. Administration of triiodothyronine to the thyroidectomized rats restored the activity to near control levels. The results suggest a role of the thyroid hormones in the regulation of glycolysis in brain.

Animals↗
PubMed1995

NADP-malate dehydrogenase activity in rat erythrocytes. Comparison with pyruvate kinase in relation to coupling with lactate dehydrogenase.

The present study explores the possible channelling of pyruvate generated by either pyruvate kinase or NADP-malate dehydrogenase to lactate dehydrogenase in cross-linked and permeabilized erythrocytes. The generation of both unlabelled and 14C-labelled pyruvate and lactate was measured in rat erythrocytes, which were prepared for cross-linking with dimethyl suberimidate and permeabilization by digitonin and then exposed to unlabelled or 14C-labelled malate and/or phospho-enol-pyruvate. Rat erythrocytes were found to display NADP-malate dehydrogenase activity. Under conditions in which the generation rates of pyruvate from either phospho-enol-pyruvate (15 microM) or malate (0.5 mM) were not vastly different from one another, a greater fraction of the 2-keto acid was converted to lactate when produced from phospho-enol-[1-14C]pyruvate rather than [U-14C]malate. This difference was most obvious when the availability of exogenous NADH was close to or somewhat below that theoretically required to ensure full conversion of endogenously formed pyruvate to lactate. These findings are compatible with the view that pyruvate generated at the pyruvate kinase level is converted to lactate more efficiently than pyruvate produced in the reaction catalysed by NADP-malate dehydrogenase.

Animals↗
PubMed1979

Apparent inhibition of pyruvate kinase by phosphocreatine and phosphoarginine.

1. Addition of a non-dialysable, heat-labile and acid-precipitable factor which was not absorbed on DEAE-cellulose column, could restore the sensitivity of the chromatographed muscle pyruvate kinase from Marphysa sanguinea towards phosphocreatine inhibition. 2. This factor, being non-specific as it acts on pyruvate kinase isozymes from different sources, demonstrated high creatine kinase activity. 3. High concentrations of ADP, creatine or replacement of ADP with IDP/UDP or high pH abolished the inhibition indicating that the inhibition was mediated through creatine kinase by depleting ADP. 4. Apparent inhibition of phosphocreatine was related to the relative activities of 3 intracellular enzymes--pyruvate kinase, creatine kinase and adenosine triphosphatase.

Animals↗
PubMed2003

Further evidence for the reliance of catalysis by rabbit muscle pyruvate kinase upon isomerization of the ternary complex between enzyme and products.

Isothermal calorimetry has been used to examine the effect of thermodynamic non-ideality on the kinetics of catalysis by rabbit muscle pyruvate kinase as the result of molecular crowding by inert cosolutes. The investigation, designed to detect substrate-mediated isomerization of pyruvate kinase, has revealed a 15% enhancement of maximal velocity by supplementation of reaction mixtures with 0.1 M proline, glycine or sorbitol. This effect of thermodynamic non-ideality implicates the existence of a substrate-induced conformational change that is governed by a minor volume decrease and a very small isomerization constant; and hence, substantiates earlier inferences that the rate-determining step in pyruvate kinase kinetics is isomerization of the ternary enzyme product complex rather than the release of products.

Animals↗
PubMedDate not supplied

[Properties of free and bound glycolytic enzymes from scorpionfish brain. III. Pyruvate kinase].

It is known that in fish tissues, in particular in brain, a part of pyruvate kinase is bound to structural elements. In this work the comparative analysis of properties of free and bound pyruvate kinase from the brain of sea teleost scorpion fish has been done. The sensitivity to change of pH medium and to heating was different in these two forms. The affinity of free enzyme for the both substrates was higher, than of the bound one although this difference was not reliable because of big individual variation. The difference of Hill coefficient in free and bound forms can indicate to different character of interaction between the active sites in oligomers of the both forms.

Animals↗
PubMed1988

Pyruvate kinase type M2 is phosphorylated at tyrosine residues in cells transformed by Rous sarcoma virus.

Chicken embryo cells (CECs) contain pyruvate kinase (PK) type M2 (M2-PK). Transformation of CECs by Rous sarcoma virus (RSV) leads to a reduction in the affinity of PK for the substrate phosphoenolpyruvate. In vitro, M2-PK can be phosphorylated at tyrosine residues by pp60v-src, the transforming protein of RSV. To study tyrosine phosphorylation of M2-PK in intact RSV-transformed cells, the protein was immunoprecipitated from 32P-labeled normal and RSV-SR-A-transformed CECs. Phosphoamino acid analysis of immunoprecipitated M2-PK revealed that M2-PK of both normal and transformed CECs contained phosphoserine and small amounts of phosphothreonine. Only M2-PK of transformed CECs contained phosphotyrosine in addition. For enzyme kinetic studies M2-PK was partially purified by chromatography upon DEAE-Sephacel and hydroxyapatite. A decreased affinity for phosphoenolpyruvate was observed 3 h after the onset of transformation using the temperature-sensitive mutant of RSV, ts-NY 68. The kinetic changes were correlated with tyrosine phosphorylation of M2-PK, but there is no direct evidence that they are caused by post-translational modification of the enzyme.

Animals↗
PubMed2002

Thermodynamics of the pyruvate kinase reaction and the reversal of glycolysis in heart and skeletal muscle.

The effect of temperature, pH, and free [Mg(2+)] on the apparent equilibrium constant of pyruvate kinase (phosphoenol transphosphorylase) (EC ) was investigated. The apparent equilibrium constant, K', for the biochemical reaction P-enolpyruvate + ADP = ATP + Pyr was defined as K' = [ATP][Pyr]/[ADP][P-enolpyruvate], where each reactant represents the sum of all the ionic and metal complexed species in M. The K' at pH 7.0, 1.0 mm free Mg(2+) and I of 0.25 m was 3.89 x 10(4) (n = 8) at 25 degrees C. The standard apparent enthalpy (DeltaH' degrees ) for the biochemical reaction was -4.31 kJmol(-1) in the direction of ATP formation. The corresponding standard apparent entropy (DeltaS' degrees ) was +73.4 J K(-1) mol(-1). The DeltaH degrees and DeltaS degrees values for the reference reaction, P-enolpyruvate(3-) + ADP(3-) + H(+) = ATP(4-) + Pyr(1-), were -6.43 kJmol(-1) and +180 J K(-1) mol(-1), respectively (5 to 38 degrees C). We examined further the mass action ratio in rat heart and skeletal muscle at rest and found that the pyruvate kinase reaction in vivo was close to equilibrium i.e. within a factor of about 3 to 6 of K' in the direction of ATP at the same pH, free [Mg(2+)], and T. We conclude that the pyruvate kinase reaction may be reversed under some conditions in vivo, a finding that challenges the long held dogma that the reaction is displaced far from equilibrium.

Animals↗
PubMed1985

Evidence for phosphorylation of pancreatic islet pyruvate kinase.

Rabbit pancreatic islet cytosol catalyzes the calcium-activated phosphorylation by [gamma 32P]ATP of a protein with a molecular weight of 57,000 that is precipitated with antipyruvate kinase antibodies. We were unable to demonstrate that phosphorylation in the presence of calcium or cAMP had any immediate effect on rat pancreatic islet pyruvate kinase activity. This finding is consistent with our inability to confirm the finding of others that pancreatic islets contain phosphoenolpyruvate carboxykinase activity (Diabetes, 34:246, 1985). Since the carboxykinase catalyzes phosphoenolpyruvate formation and pyruvate kinase catalyzes essentially the opposite reaction, if the carboxykinase were present in the beta cell, pyruvate kinase would need to be inhibited to prevent recycling of phosphoenolpyruvate.

Adenosine Triphosphate↗
PubMed1982

The effect of fructose on pyruvate kinase activity in isolated hepatocytes. Inhibition by allantoin and alanine.

1. Incubation of isolated hepatocytes with fructose at concentrations above 3 mM resulted in an apparent inhibition of pyruvate kinase assayed in crude extracts at sub-optimal phosphoenolpyruvate concentrations. 2. Fructose at concentrations below 3 mM caused an activation of the enzyme. 3. Increases in the hepatocyte contents of the positive effectors fructose 1.6-bisphosphate and fructose 1-phosphate were found at all concentrations of fructose up to 10mM. 4. Removal of the extrahepatocyte medium from the hepatocytes by washing resulted in an activation of the enzyme at all concentrations of fructose examined. 5. Inhibitors of the enzyme were shown to accumulate in the hepatocytes despite the depletion of ATP (a known negative effector) caused by higher concentrations of fructose. Indeed the inhibition of pyruvate kinase appeared to be correlated to the depletion of ATP. 6. Alanine (a known inhibitor) was shown to accumulate in hepatocytes as a consequence of incubation with fructose. 7. Allantoin and uric acid were shown to be inhibitors of a partially purified pyruvate kinase preparation assayed both in the presence and in the absence of fructose 1.6-bisphosphate. 8. Allantoin, but not uric acid, accumulated in the extrahepatocyte medium as a result of incubation of the cells with 10 mM-fructose.

Adenosine Triphosphate↗
PubMed1982

Interaction of D-phenylalanine with Co(II)-substituted rabbit muscle pyruvate kinase: kinetic and optical properties.

The kinetic and optical properties of Co(II)-substituted pyruvate kinase in the presence of D-phenylalanine (D-Phe) were investigated. The results are discussed in comparison with the effects of its optical isomer L-phenylalanine (L-Phe) on the same enzyme. The catalytic effect of D-Phe on rabbit muscle pyruvate kinase depended upon the nature of the activating divalent metal ion used. It has stimulatory effect on Mg(II)-activated enzyme, but inhibitory effect on Co(II)-activated enzyme. Unlike the inhibitory effect of L-Phe, the inhibition of Co(II)-enzyme by D-Phe was not sensitive to the changes of pH and temperature, could not be reversed by L-alanine (L-Ala), displayed hyperbolic kinetics, and was noncompetitive with respect to phosphoenopyruvate saturation. D-Phe induced substantial visible circular dichroism (CD) spectral changes of Co(II)-enzyme similar to those induced by L-Phe. Although ultraviolet CD spectrum was not affected, D-Phe induced an ultraviolet difference absorption spectral change very similar to, but much smaller than, that induced by L-Phe. Our results support that D-Phe and other amino acids interact with the enzyme at two different sites: a common site, causing similar conformational changes which bear little direct kinetic relevance, and a kinetically relevant site, which is sterically dependent upon the side chain of the amino acids.

Animals↗
PubMed1975

Pyruvate kinase from human skeletal muscle.

A simple method is described for the isolation of crystalline pyruvate kinase from human skeletal muscle. The enzyme was purified by ammonium sulfate fractionation, heat treatment and crystallization. Two crystal forms of pyruvate kinase differing in solubility but not in specific activity were found. The homogenous enzyme preparations in triethanolamine buffer, pH 7.6 reveal at 25 degrees a specific activity of 245 U per mg protein, and of 340 U/mg in potassium phosphate buffer (50 mM). The enzyme is activated by inorganic phosphate and fructosediphosphate to the same extent, and inhibited non competetively by ammonium ion. The molecular weight as measured by gel filtration is 220,000 daltons and the enzyme molecule is composed of 4 subunits.

Chromatography, Gel↗
PubMed1983

Inhibition of the pentose phosphate shunt by 2,3-diphosphoglycerate in erythrocyte pyruvate kinase deficiency.

Pentose phosphate shunt activity was studied by the release of 14CO2 from 14C-1-glucose and 14C-2-glucose in the red cells of five patients with pyruvate kinase deficiency and found to be significantly decreased after new methylene blue stimulation when compared to high reticulocyte controls. Incubated Heinz body formation was increased and the ascorbate cyanide test was positive in blood from these patients. The activity of glucose-6-phosphate dehydrogenase (G6PD) as well as that of 6-phosphogluconate dehydrogenase (6PGD) was inhibited to 20% of baseline in normal red cell haemolysate by 4 mM 2,3-diphosphoglycerate at pH 7.1. 2,3-Diphosphoglycerate was a competitive inhibitor with 6-phosphogluconate (Ki=1.05 mM) and a noncompetitive inhibitor with NADP (Ki=3.3 mM) for 6PGD. Since the intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate and NADP are below their Kms for G6PD and 6PGD, the kinetic data suggest that increased concentrations of 2,3-diphosphoglycerate in pyruvate kinase deficient red cells are sufficiently high to suppress pentose phosphate shunt activity. This suppression may be an additional factor contributing to the haemolytic anaemia of pyruvate kinase deficiency, particularly during periods of infection or metabolic stress.

2,3-Diphosphoglycerate↗
PubMed1984

Effect of 5-bromodeoxyuridine on the appearance of the liver isoenzyme of pyruvate kinase.

Hepatocyte cultures derived from 15-day foetal rats produce the liver form of pyruvate kinase (EC 2.7.1.40) only after 3 days of culture. The appearance of the liver form of the enzyme can be blocked by the addition of 5-bromodeoxyuridine on day 2 of culture, but not by addition on day 3 of culture. The reversibility of the action of 5-bromodeoxyuridine was shown when the inhibitor was added on day 2 and removed on day 4. By day 6 of culture the liver form of pyruvate kinase was detectable. The specificity of the action of 5-bromodeoxyuridine was monitored by following changes in the closely related embryonic form of the enzyme as a control. This was unaltered by the inhibitor.

Animals↗
PubMed1993

Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon.

Amino Acid Sequence↗