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PubMed1998

Six previously undescribed pyruvate kinase mutations causing enzyme deficiency.

Erythrocyte pyruvate kinase deficiency is the most common cause of hereditary nonspherocytic hemolytic anemia. We present 6 previously undescribed mutations of the PKLR gene associated with enzyme deficiency located at cDNA nt 476 G-->T (159Gly-->Val), 884 C-->T (295Ala-->Val), 943 G-->A (315Glu-->Lys), 1022 G-->A (341Gly-->Asp), 1511 G-->T (504Arg-->Leu), and 1528 C-->T (510Arg-->Ter). Two of these mutations are near the substrate binding site: the 315Glu-->Lys (943A) mutation may be involved in Mg2+ binding and 159Gly-->Val (476T) mutation has a possible effect on ADP binding. Four of six mutations produce deduced changes in the shape of the molecule. Two of these mutations, 504Arg-->Leu (1511T) and 510Arg-->Ter (1528T), are located at the interface of domains A and C. One of them (510Arg-->Ter) is a deletion of the C-terminal residues affecting the integrity of the protein. The 504Arg-->Leu mutation eliminates a stabilizing interaction between domains A and C. Changes in amino acid 341(nt 1022) from Gly to Asp cause local perturbations. The mutation 295Ala-->Val (884T) might affect the way pyruvate kinase interacts with other molecules. We review previously described mutations and conclude that there is not yet sufficient data to allow us to draw conclusions regarding genotype/phenotype relationship.

Adenosine Diphosphate↗
PubMed1988

Structure of the Aspergillus nidulans pyruvate kinase gene.

The complete nucleotide sequence of the Aspergillus nidulans pyruvate kinase gene, including its flanking sequences, is presented. The gene has a 1,578 bp coding sequence that encodes a protein of 526 amino acids; the latter is strongly homologous to the pyruvate kinases found in Saccharomyces cerevisiae (66%) and mammals (53%). The gene is interrupted by seven introns, three of which are in a conserved position compared to those present in the mammalian pyruvate kinase genes sequenced thus far. A fourth intron within the mononucleotide binding fold domain is in a conserved position with respect to the position of an intron within the NAD+ binding region of maize ADH I. The transcription start site has been determined; a major site of transcription was found 80 bp before the translation initiation codon. The promoter region of the A. nidulans pyruvate kinase gene contains no direct homologies with the TATA or CCAAT sequences in the expected region (30-70 bp) before the transcription initiation site. However, extended CT-enriched regions are found in the promotor region, similar to what has been observed in genes that are highly expressed in Saccharomyces cerevisiae and filamentous fungi.

Amino Acid Sequence↗
PubMed1985

Reaction of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine with histidine and cysteine residues in the active site of rabbit muscle pyruvate kinase.

The inactivation of rabbit muscle pyruvate kinase by 0.3 mM 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine at pH 7.8 is biphasic. The first phase proceeds rapidly to yield a partially active enzyme (46% residual activity) followed by a slower rate which leads to total inactivation. The inactivation of the first phase can be reversed by addition of 20 mM dithiothreitol, whereas the second phase is unaffected. These two phases have second-order rate constants of 250 M-1 X min-1 (dithiothreitol-sensitive reaction) and 52 M-1 X min-1 (dithiothreitol-insensitive reaction), respectively. Marked protection against inactivation is afforded by phosphoenolpyruvate and by metal-nucleotide complexes in the presence of free metal, indicating that reaction occurs in the region of the active site. Loss of approximately two sulfhydryls per enzyme subunit correlates well with the dithiothreitol-sensitive inactivation, suggesting that this phase of the inactivation may be attributable to disulfide formation. Incorporation of about one mole of fluorescent reagent per enzyme subunit correlates closely with the dithiothreitol-insensitive phase of inactivation, yielding a modified histidine residue. The quantum yield of the fluorescent sulfonylbenzoyl-1,N6-ethenoadenosine-pyruvate kinase is only 0.007, as compared to 0.54 for the parent nucleoside 1,N6-ethenoadenosine. The quenched fluorescence is consistent with stacking of the sulfonylbenzoyl moiety on the purine ring in the modified enzyme, which suggests that the altered histidine may be located in the adenine region of the metal-nucleotide binding site.

Adenosine↗
PubMed1980

Phosphorylation of human erythrocyte pyruvate kinase by soluble cyclic-AMP-dependent protein kinases. Comparison with human liver L-type enzyme.

Human red cell contain soluble adenosine-3',5'-phosphate-dependent protein kinases, which are able to phosphorylate the L' subunits of erythrocyte pyruvate kinase. Efficiency and maximum level of phosphorylation are very comparable in human liver and red cells. Phosphorylation of red cell pyruvate kinase results in the same kinetic modifications as for liver enzyme, namely a shift towards a 'T' allosteric state characterized by a decreased affinity for phosphoenolpyruvate and increased inhibition by the allosteric inhibitors ATP and alanine. In the course of red cell aging a small amount of partially proteolysed pyruvate kinase, devoid of the phosphorylatable site, appears; it resembles the subtilisin-treated L'4 enzyme and accounts for less than 20% of total pyruvate kinase subunits. Endogenous phosphorylation of pyruvate kinase from erythrocytes incubated in the presence of cyclic nucleotides produces the same kinetic modifications as phosphorylation in partially purified extract; this, however, does not change glucose consumption, lactate production and glycolytic intermediate concentrations of the incubated cells.

Cyclic AMP↗
PubMed1977

Subunit structure and hybrid formation of bovine pyruvate kinases.

After denaturing either type M or L pyruvate kinase by guanidine hydrochloride, urea, or low pH, enzymatic activity and quaternary structure can be recovered by diluting the enzyme into buffer containing beta-mercaptoethanol. After denaturation of type M pyruvate kinase by guanidine hydrochloride, the yield and polarization of the intrinsic protein fluorescence, as well as most of the circular dichroism characteristic of the native enzyme, were regained very rapidly, while enzymatic activity was recovered much more slowly. Under the conditions used, about 50% of the original M and 30-50% of the original type L activity were typically recovered. Average half-times for recovery of enzymatic activity were 37 min for type M and 104 min for type L but depended somewhat on the renaturation buffer and on protein concentrations in the renaturation medium. If types M and L pyruvate kinases are renatured together, an approximately random recombination of the two subunits types results in a five-membered hybrid set. We have used this hybridizability to determine the kinetics of reformation of the native tetramer by denaturing each isozyme and beginning its renaturation separately at various times mixing the two isozymes and continuing their renaturation together. These studies indicate that reformation of stable tetramers occurs relatively slowly, qualitatively paralleling the regain of enzymatic activity, and that tetramer formation may be necessary for enzymatic activity. Using a similar technique to test for spontaneous dissociation of the native isozymes in buffer, we find that type L, but not type M, reversibly dissociates into dimers and monomers in buffer solutions. This dissociation is decreased by the presence of the substrate, phosphoenolpyruvate, by Mg2+ ions, or by the allosteric effector, fructose bisphosphate.

Animals↗
PubMedDate not supplied

[Effect of methylguanidine and guanidinosuccinic acid on pyruvate kinase activity in human red cells].

An effect of methylguanidine and guanidinosuccinic acid on pyruvate kinase activity in human red cells was determined in vitro following a 3-hour incubation at 37 degrees C. The obtained results have shown that methylguanidine in the concentration of 1.8 x 10(-5) M/l inhibits pyruvate kinase activity by 20.8%. Pyruvate kinase activity was statistically significantly inhibited on addition of methylguanidine in the concentration of 5.4 x 10(-5) M/l whereas higher concentrations have no such an effect Guanidinesuccinic acid exerted similar but weaker effect on the activity of pyruvate kinase in human red cells. Mixture of methylguanidine (5.4 x 10(-5) m/l) and guanidinesuccinic acid (2.8 x 10(-5) M/l) does not affect pyruvate kinase activity in normal human red cells under identical experimental conditions.

Erythrocytes↗
PubMed1976

Nucleotide specificity of pyruvate kinase and phosphoenolpyruvate carboxykinase.

Various analogues of adenosine 5'-diphosphate with modifications in the heterocyclic base residue were tested as substrates of rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC. 2.7.1.40) and guinea pig liver mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32). The significance of different structural elements for the enzyme-substrate interaction is discussed. While pyruvate kinase shows a rather broad specificity for these analogues, phosphoenolpyruvate carboxykinase has a more stringent requirement for nucleotides, the intact keto and NH groups at C6 and N1 of the pyrimidine ring representing essential sites for the phosphoenolpyruvate carboxykinase substrate interaction. The biological significance of the different substrate specificities of pyruvate kinase and phosphoenolpyruvate carboxykinase is discussed as a possible metabolic control factor.

Animals↗
PubMed1990

Synergistic effects of proton and phenylalanine on the regulation of muscle pyruvate kinase.

Steady-state kinetic studies of muscle pyruvate kinase were conducted as a function of pH and phenylalanine concentrations. Results show that at a pH below 7.0, there is no observable effect of phenylalanine on the kinetic properties of muscle pyruvate kinase. When the results at a pH below 6.5 are used as the state for comparison, the kinetic results show that phenylalanine and proton exert a synergistic effect on the allosteric properties of the enzyme. A significantly greater change in Hill coefficients at high pH can be detected in the presence of phenylalanine than in its absence. To pinpoint the specific mechanism that leads to the synergistic effect, the kinetic data were resolved into the five equilibrium and two rate constants that characterize the basic two-state model. It can be shown that KTI, the binding constant of phenylalanine to the inactive T state, is strongly proton-linked. The affinity of phenylalanine for the T state increases with increasing pH. When the pH dependence of KTI was analyzed by the linked-function theory [Wyman, J. (1964) Adv. Protein Chem. 19, 224-285], it was shown that deprotonation favors phenylalanine binding to the T state. KRI (the binding constant of phenylalanine to the active R state), KTS (the binding constant of substrate to the T state), and L (the isomerization constant of the two states) not only are all weakly proton-linked but also it was shown that protonation favors the ligand-pyruvate kinase complex. KRS, the binding constant of substrate for the R state, shows no observable linkage to proton concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1993

A modified screening procedure to detect pyruvate kinase deficiency.

Beutler's screening procedure was used to detect pyruvate kinase deficiency in the local population. In this test, hemolysate and the reagent mixture are incubated and then placed at a spot on filter paper to be examined for fluorescence. Complete nonfluorescence marks the reaction endpoint, and fluorescence beyond 30 minutes indicates pyruvate kinase deficiency. It was difficult to determine this endpoint due to uneven sedimentation of unhemolyzed red cells on the spot. In this modified technique, the leukocyte-depleted red cell suspension was frozen and thawed for complete red cell lysis before being used for the test. Using both techniques, 493 health individuals and 126 anemic patients were screened for pyruvate kinase deficiency. By the conventional technique, 3.7% remained fluorescent after 30 minutes, whereas by the modified technique, none were fluorescent after 30 minutes. Quantitative assay indicated that all samples had pyruvate kinase activity levels greater than the lower limit of the reference range. We also demonstrated that blood samples from individuals with thalassemia trait were primarily responsible for the aberrant results from the conventional screening procedure.

Fluorescence↗
PubMed1994

Cloning and sequence analysis of the gene encoding pyruvate kinase in Trypanoplasma borelli.

The gene coding for pyruvate kinase in Trypanoplasma borelli has been cloned and characterized. A single gene copy was found with an open reading frame for a polypeptide of 496 amino acids and a molecular mass of 54337. The deduced amino acid sequence has a calculated net charge of -3. Comparison of the sequence with those of pyruvate kinases from members of the family Trypanosomatidae revealed amino acid identities of 58.4-61.5%, and, to some extent, conservation of residues supposed to be involved in the binding of the allosteric effector fructose 2,6-bisphosphate. Some kinetic properties of Trypanoplasma borelli pyruvate kinase have been determined and appear to be similar to those of the enzyme from Trypanosoma brucei and Leishmania mexicana.

Amino Acid Sequence↗
PubMed1988

Domain interaction in rabbit muscle pyruvate kinase. I. Effects of ligands on protein denaturation induced by guanidine hydrochloride.

The structural stability of rabbit muscle pyruvate kinase was examined. The unfolding of pyruvate kinase was induced by guanidine hydrochloride, and the process was monitored by spectroscopic techniques (fluorescence and UV absorption) and hydrodynamic measurements (sedimentation velocity, sedimentation equilibrium, densimetry, and viscometry). The spectroscopic techniques revealed that the unfolding of pyruvate kinase induced by guanidine hydrochloride is not a simple cooperative process. This suggests that different regions of pyruvate kinase are unfolding with different efficiencies in response to the denaturant. These regions are most likely related to the domain structures observed by x-ray crystallography. In the presence of L-phenylalanine, the allosteric inhibitor, the denaturation process became more cooperative, and the enzyme dissociated and unfolded at a higher denaturant concentration. The binding of phenylalanine also induced a structural change in the enzyme, rendering it more susceptible to tryptic digestion. One of the peptides, the production rate of which was increased, was isolated and sequenced. Its N terminus is located at the interface between two domains, one of which contains the active site. This evidence indicates structural changes, probably involving domain-domain interaction, for pyruvate kinase in response to phenylalanine binding.

Algorithms↗
PubMed1990

Pyruvate kinase from cytosolic fractions of the Ehrlich ascites tumour, normal mouse liver and skeletal muscle.

Comparative studies on cytosolic pyruvate kinase (PK, EC 2.7.1.40) from the Ehrlich ascites tumour, mouse liver and skeletal muscle, revealed the presence of two pyruvate kinase fractions: fraction A, salted out by ammonium sulphate between 21-30% saturation and predominant in the liver (type L); fraction B, salted out between 51-60% saturation and predominant in the tumour (type M2) or skeletal muscle (type M). The sigmoidal kinetics revealed in liver pyruvate kinase only were reconstructed in the mixture of both liver fractions A and B and characterized separately with linear kinetics in a double-reciprocal plot. L-Cysteine inhibited the neoplastic fraction B of pyruvate kinase only by decreasing its Vmax and increasing the Km values in relation to 2-phosphoenolpyruvate. Stearic acid altered kinetic parameters of both fractions A and B of pyruvate kinase from the muscle and liver, but not from the tumour. This suggests that tumours contain a pyruvate kinase variant, characterized by a greater affinity to 2-phosphoenolpyruvate as the main substrate and by a different sensitivity to low-molecular effectors, in comparison with types L, M or M2 of pyruvate kinase from normal tissues.

Ammonium Sulfate↗
PubMed1993

Comparison of pyruvate kinase variants from rat liver and Morris hepatoma 7777, obtained by an affinity chromatography on blue sepharose CL-6B.

Fractions A (salted out by ammonium sulphate between 21-30% saturation), and fractions B (salted out between 51-70% saturation) of pyruvate kinase (EC 2.7.1.40.) corresponding respectively to pyruvate kinase types L and M2 from rat liver and Morris hepatoma 7777 were purified by an affinity chromatography on Blue Sepharose CL-6B. Peaks of inactive proteins were eliminated and the enzyme fractions bound biospecifically to the gels were eluted by free ADP. The molecular mass of purified hepatoma pyruvate kinase fraction B was smaller than that of liver pyruvate kinase fraction B. Morris hepatoma pyruvate kinase fraction B represented a variant of type M2, characterised by greatest affinity to 2-phosphoenolpyruvate as a main substrate and different sensitivity to low-molecular effectors in comparison with types L from both liver and hepatoma and in comparison with type M2 from normal rat liver. Only this hepatoma fraction B showed a tumour specific sensitivity to L-cysteine and was insensitive to normal signal molecules i.e. to ATP and fructose-1,6-diphosphate which influence liver pyruvate kinase activity. L-Cysteine inhibited the tumour fraction B of pyruvate kinase by decreasing its Vmax and increasing the Km values in relation to 2-phosphoenolpyruvate.

Animals↗
PubMed1984

Pyruvate kinase activity in isolated rat hepatocytes during a feeding cycle and during fasting.

The short-term regulation of pyruvate kinase in rat hepatocytes was studied during a feeding cycle or progressive fasting. In fed and fasted rats, the activity ratio of pyruvate kinase (V0.5-mMPEP/Vmax) in crude extracts was directly correlated with the concentration of glucose 1,6-bisphosphate++ in hepatocytes. Precipitation of the enzyme from homogenates with ammonium sulphate, which removes fructose 1,6-bisphosphate, induced in both groups of animals a low activity ratio of pyruvate kinase which remained unchanged during the whole experiment. These results show that in absence of added glucagon in hepatocytes, the activity of pyruvate kinase is mainly controlled by the intracellular level of fructose 1,6-bisphosphate. Addition of glucagon to hepatocytes from fed or fasted rats inactivated pyruvate kinase and decreased the concentration of fructose 1,6-bisphosphate in cells. However, in crude extracts both the activity ratio of pyruvate kinase and fructose 1,6-biphosphate levels were higher in fed rats than in starved rats. These findings suggest that glucagon-induced inactivation of pyruvate kinase also depends upon the concentration of fructose 1,6-biphosphate in hepatocytes.

Animals↗
PubMed1982

Insulin mediates the stimulation of pyruvate kinase by a dual mechanism.

A radioimmunoassay specific for liver pyruvate kinase was used to determine the mechanism(s) involved in the insulin stimulation of this enzyme activity in chronically diabetic rats. Rats, made diabetic with alloxan, were fed on a high-carbohydrate (50%-sucrose) fat-free diet and treated with insulin for 12, 36 or 60 h. Livers were removed at the various times, a piece was kept for determination of glycogen, and the remainder was homogenized. The 100000 g supernatant was prepared and used for determination of pyruvate kinase activity and quantity. Glycogen increased to a maximum of approx. 7% by 12 h after insulin treatment, and was maintained at this elevated value for 60 h. Liver pyruvate kinase activity, which is depressed in diabetes, did not respond to insulin until 36 h of treatment, with a more substantial increase occurring by 60 h. Radioimmunoassay data indicated that the increase in activity was concomitant with a substantial increase in the quantity of the enzyme and a moderate increase in its specific activity. These results demonstrate that a dual mechanism, i.e. an increase in both the quantity and specific activity of the enzyme, regulates the insulin-mediated stimulation of liver pyruvate kinase in the diabetic rat.

Animals↗
PubMed2001

Human erythrocyte pyruvate kinase: characterization of the recombinant enzyme and a mutant form (R510Q) causing nonspherocytic hemolytic anemia.

Human erythrocyte pyruvate kinase plays an important role in erythrocyte metabolism. Mutation on the gene results in pyruvate kinase deficiency and is an important cause of hereditary nonspherocytic hemolytic anemia. Because of difficulties in isolating the mutant enzymes from patients, these mutations have not been fully studied. In this study, a complementary DNA (cDNA) encoding the human erythrocyte pyruvate kinase was generated. The cDNA was cloned into several expression vectors, and the protein was expressed and purified. The tetrameric protein exhibited properties characteristic of authentic human erythrocyte pyruvate kinase, including response to substrate, phosphoenolpyruvate, activation by fructose 1,6-bisphosphate, and inhibition by adenosine triphosphate (ATP). The N-terminal segment of the protein was highly susceptible to proteolysis, but only 2 of the 4 subunits were cleaved and lacked 47 N-terminal amino acid residues. A mutant protein, R510Q, which is the most frequently occurring mutation among Northern European population, was also generated and purified. The mutant protein retained its binding capacity to and could be activated by fructose 1,6-bisphosphate and showed similar kinetics toward phosphoenolpyruvate and adenosine diphosphate as for the wild-type enzyme. Conversely, the mutant protein has a dramatically decreased stability toward heat and is more susceptible to ATP inhibition. The enzyme instability decreases the enzyme level in the cell, accounting for the clinically observed "pyruvate kinase deficiency" of patients who are homozygous for this mutation. This study provides the first detailed functional characterization of human erythrocyte pyruvate kinase. These findings will allow the establishment of a fine correlation between molecular abnormalities and the clinical expression of the disease.

Adenosine Triphosphate↗
PubMed1988

[Kinetic model of the functioning of pyruvate kinase from bovine adrenal cortex].

The dependence of pyruvate kinase reaction rate on the concentration of one of the ligands--ADP or MgCl2--at constant concentrations of the other ligand was studied. The enzyme activity vs ligand concentration curves have fairly symmetrical peaks which correspond to the range of approximately equal ligand concentrations. The S-shaped dependence is observed only over the range of concentrations close to the dissociation constant for the Mg-ADP- complex (0.7 mM) under the given experimental conditions. The data obtained are consistent with the results of the first model kinetics within the framework of the London-Steck theory. The substrate for pyruvate kinase is the Mg-ADP- complex, while free Mg2+ and ADP3- competitively inhibit the enzyme. The inhibition constants are equal to 44 and 1 mM, respectively. The inhibiting effects of the metal and dinucleotide may be due to the competition with the substrate for the enzyme active site. Taking into consideration the fact that the binding of one of the ligands to the enzyme depends on the presence of the other ligand, a conclusion is drawn that Mg2+ forms a bridge with ADP3- and pyruvate kinase from adrenal cortex.

Adrenal Cortex↗
PubMed1987

Dietary and hormonal regulation of L-type pyruvate kinase gene expression in rat small intestine.

L-type pyruvate kinase is an enzyme of the glycolytic pathway whose activity and mRNA levels fluctuate in the small intestine according to dietary status. Both the enzyme activity and mRNA concentration decline during fasting and increase upon refeeding either a glucose-rich or a fructose-rich diet. Using a single-strand M 13 phage complementary to L-type pyruvate kinase mRNA as probe, we determined the level of the mRNA in the small intestine of normal, adrenalectomized, thyroidectomized, diabetic and glucagon-treated or cAMP-treated animals refed either a glucose-rich or a fructose-rich diet. The specific mRNA is present in the small intestine of normal fasted rats and increases twofold and threefold on refeeding glucose and fructose respectively. However, the hormonal control of the gene expression differs according to the dietary carbohydrate. The L-type pyruvate kinase mRNA increase, induced by glucose feeding, is hormone-dependent and requires the presence of thyroid hormones and insulin. In fructose-fed rats a certain level of mRNA increase occurs regardless of the hormonal status of the animals, but the full induction of the mRNA by fructose requires the presence of glucocorticoids, thyroid hormones and insulin. Thus, the hormonal regulation of L-type pyruvate kinase gene expression in the small intestine is largely similar to that described in normal rat liver but the basal mRNA level and the stimulation of the mRNA increase by fructose are higher in the small intestine.

Adrenalectomy↗