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PubMed2000

Leishmania pyruvate kinase: the crystal structure reveals the structural basis of its unique regulatory properties.

Glycolysis occupies a central role in cellular metabolism, and is of particular importance for the catabolic production of ATP in protozoan parasites such as Leishmania and Trypanosoma. In these organisms pyruvate kinase plays a key regulatory role, and is unique in responding to fructose 2,6-bisphosphate as allosteric activator. The determination of the crystal structure of the first eukaryotic pyruvate kinase in the T-state (the inactive or 'tense' conformation of allosteric enzymes) is described. A comparison of the effector sites of the Leishmania and yeast enzymes reveals the structural basis for the different effector specificity. Two loops, comprising residues 443-453 and 480-489, adopt very different conformations in the two enzymes, and Lys-453 and His-480 that are a feature of trypanosomatid enzymes provide probable ligands for the 2-phospho group of the effector molecule. These and other differences offer an opportunity for the design of drugs that would exploit regulatory differences between parasite and host.

Amino Acid Sequence↗
PubMed1990

[Chronic non-spherocytic hemolytic anemia caused by pyruvate kinase deficiency in a Costa Rican family carrying hemoglobin C disease].

The two first homozygous (or double heterozygous) cases of pyruvate kinase (PK) deficiency found in a Costa Rica family with no signs of consanguinity are reported. The clinical manifestations of the deficiency were present in both cases, these being enhanced in one of them by pregnancy. The family study performed showed the heterozygous character of the PK deficiency in all cases, plus the demonstration in two instances (father and brother) of a heterozygous haemoglobin C disease. The importance of the PK/HK quotient in the identification of the PK deficiency heterozygous is stressed, especially when the enzyme activity registered from haemolysates falls within the normal range.

Adult↗
PubMed1980

Characterization and kinetics of isoenzymes of pyruvate kinase from developing castor bean endosperm.

Isozymes of pyruvate kinase (PK) have been isolated from developing castor bean endosperm. One isozyme, PK(c), is localized in the cytosol, and the other, PK(p), is in the plastid. Both isozymes need monovalent and divalent cations for activity, requirements which can be filled by K(+) and Mg(2+). Both isozymes are inhibited by citrate, pyruvate, and ATP. PK(c) has a much broader pH profile than PK(p) and is also more stable. Both have the same K(m) (0.05 millimolar) for PEP, but PK(p) has a 10-fold higher K(m) (0.3 millimolar) for ADP than PK(c) (0.03 millimolar). PK(c) also has a higher affinity for alternate nucleotide substrates than PK(p). The two isozymes have different kinetic mechanisms. Both have an ordered sequential mechanism and bind phosphoenolpyruvate before ADP. However, the plastid isozyme releases ATP first, whereas pyruvate is the first product released from the cytosolic enzyme. The properties of the two isozymes are similar to those of their counterparts in green tissue.

Journal Article↗
PubMed1993

The induction of pyruvate kinase synthesis by heat shock in Xenopus laevis embryos.

Heat-shocked Xenopus embryos have an unusually complex heat shock response. The dominant heat shock protein (Hsp) has a relative molecular mass (M(r)) of 62,000 D (Hsp62). Affinity-purified IgGs against the glycolytic enzyme pyruvate kinase (PK; EC 2.7.1.40) specifically immunoprecipitated Hsp62 from extracts of embryos that had been heat-shocked at 37 degrees C for 30 min. Thus, Hsp62 and pyruvate kinase are immunologically cross-reacting. Electrophoretic separation of PK isoforms suggests that heat-shocked Xenopus embryos increase synthesis of an isoform of PK. Thermal denaturation studies suggest that this isoform has enhanced thermal stability. The identification of PK as an Hsp is discussed within the context of a physiological requirement for elevated levels of anaerobic glycolysis in heat-stressed cells as a vital component of the acquisition of thermotolerance.

Animals↗
PubMed1990

Endotoxin, epinephrine, glucagon, insulin and calcium ionophore A23187 modulation of pyruvate kinase activity in cultured rat hepatocytes.

Altered glucose metabolism is one of the commonly observed sequelae of sepsis and septic shock. The present investigation was undertaken to determine the role of endotoxin (ET) upon hepatocyte glucoregulation, by measuring the activity of pyruvate kinase (PK), a key glycolytic enzyme. Hepatocytes were exposed to endotoxin concentrations known to occur in vivo during sepsis, i.e., from 1 X 10(-14) to 1 X 10(-8) g/ml. The alteration of the enzyme activities after addition of epinephrine, glucagon, insulin and calcium ionophore A23187 with and without ET preincubation were also examined. ET alone decreased the PK activity by 12% at all concentrations tested. The basal inhibition of the enzyme caused by epinephrine (-48%) was partially blocked by ET preincubation above 1 X 10(-10) g/ml. There were no ET-(glucagon, calcium ionophore, insulin) interaction. These in vitro results do not support pyruvate kinase as a site of hepatic enzyme regulation defect in endotoxaemia.

Animals↗
PubMed1985

Dephosphorylation of L-pyruvate kinase during rat liver hepatocyte isolation.

In isolated rat liver cells, the inhibition of L-pyruvate kinase (L-PK) by a cyclic AMP-dependent phosphorylation mechanism is involved in the hormonal control of glycolysis and gluconeogenesis. The aim of this study was to ascertain whether or not the in vivo phosphorylation state of the enzyme was maintained during the liver perfusion used to prepare isolated liver cells. When the L-PK phosphorylation state was studied indirectly in liver extracts by kinetic measurement, it was found that, during the perfusion, the S0.5 of phosphoenol pyruvate (PEP) for L-PK was decreased in a time-dependent manner from 1 +/- 0.08 to 0.64 +/- 0.1 mM (P less than 0.01) and 0.58 +/- 0.06 mM in liver cells. This shift was prevented only by the addition of glucagon to the perfusion medium. The extent of phosphorylation of L-PK was also estimated by incubation of the liver extract with [gamma-32P]ATP, protein kinase, and cyclic AMP, and measurement of 32Pi incorporated in L-PK by specific immunoprecipitation. In liver extracts removed at the beginning of the perfusion, 0.4 mol Pi/mol L-PK was incorporated and there was no stimulation by cyclic AMP. In contrast, in the liver extracts removed after 30 min of perfusion, cyclic AMP stimulated 32P incorporation two to threefold, and 1.6 mol Pi/mol L-PK was incorporated. These data suggest that L-PK was activated by a dephosphorylation mechanism during rat liver perfusion. This phenomenon could be involved in the classical inactivation of gluconeogenesis observed in the perfused rat liver model.

Animals↗
PubMed1974

A kinetic study of pig liver pyruvate kinase activated by fructose diphosphate.

The paper reports a study of the reaction between phosphoenolpyruvate, ADP and Mg(2+) catalysed by pig liver pyruvate kinase when activated by fructose diphosphate and K(+). The experimental results are consistent with two non-sequential mechanisms in which the substrates and products of the reaction are phosphoenolpyruvate, ADP, Mg(2+), pyruvate and MgATP. Pyruvate release occurs before ADP binding. Two Mg(2+) ions are involved, though the two Mg(2+)-binding sites cannot be occupied simultaneously. An isomerized enzyme complex forms before release of MgATP. Values were determined for the Michaelis constants of the reaction. Apparent MgATP inhibition constants are also given.

Adenosine Diphosphate↗
PubMed1976

Arrangement and conformations of substrates at the active site of pyruvate kinase from model building studies based on magnetic resonance data.

Seventeen distances from two paramagnetic reference points, as determined by nuclear relaxation studies of six active complexes of rabbit muscle pyruvate kinase, have been used to construct molecular models of two composite enzyme complexes. In the model of the hypothetical pyruvate kinase-M(I)-M(II)-ATP-Cr(III)-P-enolpyruvate complex, overlap of the transferred phosphoryl groups of the two substrates, which is required to explain the observed competition, is incomplete, allowing greater than or equal to 1 A for the transition state to form. In the active enzyme-M(I)-M(II)-ATP-Cr(III)-pyruvate complex, the gamma-phosphoryl phosphorus of ATP is in molecular contact (3.0 +/- 0.5 A) with the carbonyl oxygen of pyruvate, consistent with direct phosphoryl transfer, indicating no need for intermediate phosphorylation of the enzyme. The enzyme-bound divalent cation, which forms second sphere complexes with the phosphoryl groups of P-enolpyruvate and ATP, may activate the transferred phosphoryl group indirectly, through a water ligand. By analogy with the position of Cr(III), a second divalent cation may participate more directly by coordination of the triphosphate chain of ATP.

Adenosine Triphosphate↗
PubMed1999

A proposal for the locus of metformin's clinical action: potentiation of the activation of pyruvate kinase by fructose-1,6-diphosphate.

Reduction of hepatic glucose output has been shown to be the chief basis for metformin's clinical benefit in diabetes, and the balance of the evidence suggests that this reflects inhibition of gluconeogenesis. Recent research with hepatocyte cell cultures demonstrates increased flux through pyruvate kinase in metformin-treated cells. An analysis of the conditions under which clinically relevant concentrations of metformin inhibit gluconeogenesis in hepatocyte cultures prompts the hypothesis that metformin potentiates the allosteric activation of pyruvate kinase by fructose-1,6-diphosphate. This model rationalizes several salient features of metformin's clinical activity: its ability to reduce hepatic triglyceride synthesis, its appetite-suppressant effect, and its failure to induce hypoglycemia.

Diabetes Mellitus↗
PubMed2004

Unexpectedly high but still asymptomatic iron overload in a patient with pyruvate kinase deficiency.

Iron overload is a serious condition, which may lead to irreversible organ damage. The risk of iron accumulation in pyruvate kinase deficiency (PKD) has traditionally been regarded as low, but recent evidence has questioned this notion. We here present a case of a young PKD patient showing evidence of asymptomatic iron accumulation measured as liver iron concentration (LIC) obtained noninvasively by magnetic resonance imaging. The iron overload was not related to blood transfusions, but rather secondary to concomitant risk factors leading to increased intestinal iron absorption, such as chronic hemolysis and splenectomy. The iron status of PKD patients, preferably assessed by LIC measurements, should therefore be evaluated regularly also in asymptomatic patients. This evaluation should start already at a young age, in order to initiate iron chelation before the development of iron-induced organ damage.

Adult↗
PubMed1987

A phospho-oligosaccharide mimics insulin action on glycogen phosphorylase and pyruvate kinase activities in isolated rat hepatocytes.

A phospho-oligosaccharide which is the polar head group of a novel insulin-sensitive glycophospholipid has recently been involved in insulin action. We have investigated the insulin-like effects of this phospho-oligosaccharide on both glycogen phosphorylase a and pyruvate kinase activities of hepatocytes incubated in the presence of glucagon (0.1 nM). Similarly to insulin, the phospho-oligosaccharide antagonized glucagon-dependent activation of glycogen phosphorylase, as well as the inactivation of pyruvate kinase caused by this hormone. The antagonistic action of the phospho-oligosaccharide on glucagon effects was dose-dependent. Furthermore, it partially antagonized glucagon-stimulated cyclic AMP levels. These results support the hypothesis that this phospho-oligosaccharide mediates at least some insulin actions in hepatocytes.

Animals↗
PubMed1969

Inhibition of human brain pyruvate kinase and hexokinase by phenylalanine and phenylpyruvate: possible relevance to phenylketonuric brain damage.

In phenylketonuria high levels of L-phenylalanine are present along with increased levels of phenylpyruvic acid. The present work shows that L-phenylalanine is a competitive inhibitor of human brain pyruvate kinase and phenylpyruvic acid is an inhibitor of human brain hexokinase. The enzymes have approximately the same K(i) for these inhibitors in adult and in fetal human brain. However, in the fetal human brain the absolute activities for both enzymes are less than 10 per cent of those found in the adult. Thus, the fetal brain enzymes may be more vulnerable to inhibition by these compounds in the phenylketonurics. The inhibition of human brain pyruvate kinase and hexokinase by L-phenylalanine and phenylpyruvic acid may have a role in the brain damage in phenylketonurics.

Aging↗
PubMed1983

Primary structure of chicken muscle pyruvate kinase mRNA.

We have determined the cDNA sequence corresponding to chicken muscle pyruvate kinase mRNA; the predicted coding region spans 529 amino acids and establishes the complete amino acid sequence for the vertebrate enzyme. We demonstrate that the level of mRNA for this enzyme is under developmental control and suggest a structural model for the protein kinase-mediated regulation of the mammalian liver isozyme. We report a method for the direct analysis of, and the preparation of cDNA probes from, mRNA which has been fractionated on methylmercury/agarose gels.

Amino Acid Sequence↗
PubMed1979

Rat hyperplastic hepatic nodules and primary hepatomas retaining abnormally high pyruvate kinase L type activity.

As reported in our previous paper (Sato et al., Cancer Res., 38: 3086-3093, 1978), most of the hyperplastic hepatic nodules and primary hepatomas induced by N-2-fluorenylacetamide (2-FAA), diethylnitrosamine (DENA), and 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) showed that pyruvate kinase liver (L) type decreases and the prototypic M2 type increases concomitantly with dedifferentiation of tissues. However, at least 14 samples among 120, mostly hyperplastic nodules and highly differentiated hepatomas induced by 2-FAA or DENA, retained exceptionally high activities of the L type, while other enzymes of carbohydrate metabolism in these tissues deviated toward a common pattern similar to that in the fetal liver. Individual patterns of 9 enzymes including pyruvate kinase of carbohydrate metabolism in these samples are arranged and discussed as examples of unbalanced enzyme deviation in hepatocarcinogenesis.

Animals↗
PubMed1980

Iron overload in congenital erythrocyte pyruvate kinase deficiency.

Severe iron overload developed in two patients with congenital erythrocyte pyruvate kinase deficiency. In one of them this was complicated by hepatic cirrhosis and endocrine dysfunction. In both patients, the institution of intermittent subcutaneous administration of desferrioxamine resulted in significant urinary iron excretion and a reduction in the degree of iron overload.

Adult↗
PubMed1977

The effect of proflavine on pyruvate kinase I of Escherichia coli B.

Proflavine (PF) inhibited glucose use in sensitive but not resistant Escherichia coli B. Glucose transport (as measured by alpha-methylglucoside accumulation) was only partly inhibited by PF concentration that completely blocked glucose use. Fructose 1,6-diphosphate-(FDP)-regulated pyruvate kinase (PK1) (EC 2.7.1.40), the only glycolytic enzyme affected by PF, was completely inhibited by a dye concentration of 0.8 mM. The inhibition curve for PF was sigmoidal, suggesting that PF was acting as an allosteric inhibitor. PF increased the K 1/2 for phosphoenolpyruvate (PEP) and lowered the V; however, it had no effect on the Hill number for PEP. PF inhibition was partially reversed by FDP but not by cyclic AMP, AMP, ATP, fuctose 6-phosphate, or dithiothreitol. Studies with a variety of acridines indicated that those substituted at the 3-position are the most effective inhibitors and also that hydrophobic interactions may be involved in PF inhibition of PK I. PK I for E. coli B/Pr was also strongly inhibited by PF, indicating that PF resistance does not lie at the level of this enzyme. Ribose-5-phosphate-regulated pyruvate kinase (EC 2.7.1.40) was much less sensitive that PK I to the inhibitory effects of PF. A role for PF as a molecular probe for PK I has been proposed.

Acridines↗
PubMedDate not supplied

A family case of pyruvate kinase variant, PK 'Nichinan'.

A case of congenital hemolytic anemia caused by pyruvate kinase (PK) deficiency variant designated PK 'Nichinan' is presented. A 29-old patient with a history of anemia and hepatosplenomegaly had an episode of aplastic crisis associated with fever, abdominal symptoms and worsening hepatosplenomegaly. Study of the family members revealed that his mother and two of three siblings showed decreased PK activity whereas his father showed normal PK activity with a normal level of glycolytic intermediates. The patient was assumed to be a double heterozygote with two separate mutant genes from the parents despite the apparent normality of his father's erythrocyte PK.

Adult↗
PubMed1984

Molecular and cellular regulation of pyruvate kinase in red blood cells.

The main sources of the heterogeneity of pyruvate kinase (PK) isoenzymes are discussed. The regulative role of the L-type PK for the energy metabolism of a tissue without glyconeogenesis has been examined in erythrocytes. For that purpose a kinetic model of the PK has been established taking into account the substrates and the effectors ATP and fructose-1,6.biphosphate. This PK model was incorporated into a complex mathematical model of glycolysis and the concentrations of metabolites and the glycolytic flux were calculated for normal PK activity and for a PK activity ten times lower than normal. From calculations it is concluded that under the conditions of PK deficiency the energy metabolism cannot be stabilized. This corresponds to the experimental finding that the filtration constant of PK deficient red blood cells increases with declining survival time.

Erythrocytes↗