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PubMed1990

Determination of the catalytic pathway of C4-leaf pyruvate, orthophosphate dikinase from maize.

The mechanism of the maize pyruvate, phosphate dikinase-catalyzed conversion of adenosine 5'-triphosphate, orthophosphate and pyruvate to adenosine 5'-monophosphate, inorganic pyrophosphate and phosphoenolpyruvate, respectively, was determined by using transient kinetic techniques. The data obtained demonstrate that catalysis in the maize pyruvate, phosphate dikinase active site involves initial transfer of the P beta P-unit from adenosine 5'-trisphosphate to the enzyme to form a pyrophosphorylenzyme intermediate, followed by sequential phosphoryl group transfer to orthophosphate (to form pyrophosphate and a phosphoenzyme intermediate) and pyruvate (to form phosphoenolpyruvate and free enzyme).

Kinetics↗
PubMed1992

Effects of substitution of aspartate-440 and tryptophan-487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis.

A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site-directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half-saturating concentrations increasing from 0.64 to 9.0 microM for thiamin diphosphate and from 4.21 to 45 microM for Mg2+. Unlike the wild-type enzyme, there was little quenching of tryptophan fluorescence upon adding cofactors to this modified form. The data suggest that tryptophan-487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine or glycine for aspartate-440, a residue which is conserved between many thiamin diphosphate-dependent enzymes, completely abolishes enzyme activity.

Amino Acid Sequence↗
PubMed1983

The immunological and catalytically active form of L-type pyruvate kinase in rat liver cytosol.

The protein species precipitated from rat liver cytosol by rabbit antisera raised to pure L-type pyruvate kinase were investigated by sodium dodecyl sulphate gel electrophoresis. The primary antisera (anti-L-type pyruvate kinase) precipitated protein species with mol. wts 56,000, 41,000 and 39,000. The 41,000 mol. wt protein was identified as fructose-bis-phosphatase. Double diffusion and immunotitration experiments established that L-type pyruvate kinase and fructose-bis-phosphatase shared common antigenic determinants. This information enabled an improved antiserum (anti-LPK) to be obtained. The use of anti-LPK showed that the 56,000 mol. wt subunit was the only catalytically and immunologically active form of L-type pyruvate kinase in liver. This was confirmed by biosynthetic experiments with cultured hepatocytes. The specific activity of the enzyme in liver extracts was also determined by quantitative immunotitration with anti-LPK. Despite changes in dietary status which varied the concentration of enzyme protein, the maximum specific activity of the enzyme remained constant and essentially the same as that of pure enzyme.

Animals↗
PubMed1983

An approach to the elucidation of the quaternary structure role in the activity of pyruvate kinase studies on the immobilized enzyme.

1. Studies on pyruvate kinase immobilized via -S-S- linkages showed that single subunits of this tetrameric enzyme are inactive. 2. Pyruvate kinase was coupled with different preparations of CNBr-activated Sepharose via one and more than one bond, and after removal of non-covalently bound subunits by denaturant treatment the subunit derivatives were obtained composed of different pyruvate kinase species. 3. Heat inactivation studies of immobilized enzyme derivatives suggested that not only tetramers of pyruvate kinase are active. 4. This idea is further supported by experiments with 2 M urea dissociation of immobilized tetrameric enzyme carried out in the presence of Mg2+ ions.

Animals↗
PubMed1985

The effect of ethanol or sorbitol on glucose production from pyruvate in isolated hepatocytes from 48-hour fasted guinea-pigs.

Hepatocytes isolated from 48-hour, fasted guinea-pigs were incubated with glucose precursors to compare relative rates of glucose production. Glucose production from lactate and pyruvate was similar (2.61 vs 3.18 mumol/hr per 100 mg wet weight). Glucose production from fructose was greater than that from sorbitol (4.68 vs 1.63 mumol/hr per 100 mg wet weight). When ethanol was added to pyruvate-containing buffer, the flux of pyruvate to glucose and lactate was synergistically enhanced (5.28 vs 3.76 and 7.51 vs 2.88 mumol/hr per 100 mg wet weight, respectively). When sorbitol was added to buffer containing pyruvate, glucose and lactate production were even greater than that seen with ethanol (8.32 vs 5.38 and 15.99 vs 7.51 mumol/hr per 100 mg wet weight, respectively).

Animals↗
PubMed1987

Liver specific pyruvate kinase in pheasants Phasianus colchicus.

While in chickens, the existence of a liver form of pyruvate kinase is controversial, the liver form of pyruvate kinase in pheasants, murres and puffins is electrophoretically distinct from that in muscle, brain, kidney, lung and small intestine. Although the forms in lungs, muscle, heart, brain and small intestine could not be reliably separated by electrophoresis, the functional characteristics of the lung and muscle forms of pyruvate kinase in the pheasant are distinct and can be classified as K and M isozymes respectively. Our data suggest that these birds possess at least three distinct isozymes of pyruvate kinase.

Animals↗
PubMed1988

Regulation of pyruvate kinase in Reuber H35 hepatoma cells by insulin and fructose.

1. Kinetic and immunological studies as well as electrophoretic behaviour indicated that pyruvate kinase in Reuber H35 hepatoma cells is of the M2-type. 2. Addition of 0.1 microM insulin or 2 mM fructose to the incubation medium for 72 hr increased the activity of the M2-type pyruvate kinase in Reuber H35 hepatoma cells by 103 and 25% respectively. 3. Incorporation studies with [3H]leucine followed by immunoprecipitation showed that the apparent rate of synthesis of the M2-type pyruvate kinase was increased by both insulin and fructose. 4. Degradation studies indicated that the addition of insulin and fructose to the incubation medium increased the half-life of the M2-type pyruvate kinase from 4.8 to 8.6 and 6.8 hr respectively.

Animals↗
PubMed1990

Application of high-performance liquid chromatography to the purification, disintegration and molecular mass determination of pyruvate dehydrogenase multi-enzyme complexes from different sources.

The pyruvate dehydrogenase complex is associated with the inner mitochondrial membrane. A gentle and rapid purification procedure, especially for the very unstable pyruvate dehydrogenase complex from the extremely thermophilic organism Thermus aquaticus, is described. This procedure is based essentially on a combination of hydrophobic interaction and of adsorption chromatography by the rapid fast protein liquid chromatographic technique. Applying the same method, a relative molecular mass of 9.1 . 10(6) daltons was obtained by gel filtration on Superose 6 HR 10/30 for the pyruvate dehydrogenase complex from T. aquaticus. The same column served to resolve the pyruvate dehydrogenase complex into its enzyme components.

Animals↗
PubMed1985

Distribution of phosphate-activated glutaminase, succinic dehydrogenase, pyruvate dehydrogenase and gamma-glutamyl transpeptidase in post-mortem brain from Huntington's disease and agonal cases.

The activity of phosphate-activated glutaminase was reduced throughout the brain of cases with longstanding illnesses (agonal controls) compared to cases dying suddenly. The reduction was less marked in cortical than sub-cortical areas, with the caudate nucleus occupying an intermediate position. In control brains succinic dehydrogenase and pyruvate dehydrogenase were little affected by the ante-mortem clinical state. Of 9 brain areas studied, only the caudate nucleus showed a reduction of phosphate-activated glutaminase and succinic dehydrogenase in Huntington's disease greater than in agonal controls. The levels of succinic dehydrogenase and pyruvate dehydrogenase were highly correlated in frontal cortex and in caudate nucleus of Huntington's disease and control brains. There was a significant reduction in pyruvate dehydrogenase mean activity and a significant increase in gamma-glutamyl transpeptidase mean activity in Huntington's disease caudate nucleus. The level of pyruvate dehydrogenase significantly decreased and the level of gamma-glutamyl transpeptidase significantly increased with increasing duration of illness, possibly due to a progressive loss of neurons and increase in the density of glia in Huntington's disease caudate nucleus.

Adult↗
PubMed1989

Familial intermittent ataxia due to a defect of the E1 component of pyruvate dehydrogenase complex.

Disturbances of pyruvate metabolism have been implicated in the aetiology of several neurological disorders including Leigh's disease and familial ataxia. We have re-investigated a patient whose initial description documented intermittent ataxia, a presumed disorder of pyruvate metabolism and an X-linked pattern of inheritance. Recent studies showed he had slow oxidation of pyruvate, low pyruvate dehydrogenase complex (PDC) activity and immunochemical evidence of E1 deficiency in skeletal muscle mitochondria. This is consistent with the recent finding that the gene for E1 alpha is on the X chromosome.

Adult↗
PubMed1983

The effects of cyclopropane carboxylic acid on hepatic pyruvate metabolism.

The effects of the hypoglycemic agent, cyclopropane carboxylate, on the metabolism of various substrates that enter the mitochondrion via the mitochondrial monocarboxylate transporter were investigated in perfused rat livers. Metabolism of pyruvate, branched-chain alpha-keto acids, acetoacetate and, to a lesser extent beta-hydroxybutyrate, were all inhibited by cyclopropane carboxylate. In addition, the stimulation of pyruvate decarboxylation by beta-hydroxybutyrate at low pyruvate concentrations because of exchange of extramitochondrial pyruvate with intramitochondrially generated acetoacetate, was abolished by cyclopropane carboxylate. Gluconeogenesis from alanine, which is not transported via the monocarboxylate translocator, was not inhibited by low concentrations of cyclopropane carboxylate. Cyclopropane carboxylate also inhibited fatty acid oxidation, as measured by ketone body production. These results support previous findings that the hypoglycemic agent, cyclopropane carboxylate, exerts at least some of its metabolic effects at the level of the mitochondrial monocarboxylate transporter.

Animals↗
PubMed1985

Intron/exon structure of the chicken pyruvate kinase gene.

The chicken pyruvate kinase gene is interrupted by at least ten introns, including nine introns within the coding region. We compare the structure of this gene with the three-dimensional protein structure of the homologous cat muscle enzyme. The introns are not randomly placed--they divide the coding sequence into fairly uniformly sized pieces encoding discrete elements of secondary structure. The introns tend to fall at interruptions between stretches of alpha-helix or beta-sheet residues, and each of the six exons that contribute to the barrel-shaped central domain include one or two repeats of a simple unit, an alpha-helix plus a beta strand. This structure suggests that introns were not inserted into a previously uninterrupted coding sequence, but instead are products of the evolution of the first pyruvate kinase gene. We have found some sequence homology between a segment of pyruvate kinase and the structurally homologous mononucleotide binding fold of alcohol dehydrogenase. The superposition of these two regions aligns an intron from the maize alcohol dehydrogenase gene four nucleotides from an intron in the chicken pyruvate kinase gene.

Amino Acid Sequence↗
PubMed1988

Purification and characterization of a metabolite-regulated pyruvate kinase from Leishmania major promastigotes.

The pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) of Leishmania major promastigotes is a multimer of 59 kDa subunits having an Mr 181000. It is activated by its substrate phosphoenolpyruvate (PEP) in a positively cooperative manner, and heterotropically by fructose 1,6-bisphosphate (FBP). Kinetics with regard to the phosphate acceptor adenosine 5'-diphosphate (ADP), MgCl2, and KCl are hyperbolic and unaffected by FBP. The enzyme is strongly inhibited by the reaction product ATP, as well as GTP and ITP, and to a lesser degree by citrate. Of seven amino acids reported to inhibit the pyruvate kinases of other organisms, none have any effect on the L. major pyruvate kinase in vitro. The enzyme shows its maximum activity at pH 7.0 in the absence of FBP, and at pH 7.6 in its presence. Contrary to previous suggestions, the enzyme appears to be well-suited for a regulatory role in the metabolism of an aerobic organism capable of net glucose synthesis.

Animals↗
PubMed1996

Characterisation and purification of pyruvate:ferredoxin oxidoreductase from Giardia duodenalis.

The major 2-oxoacid oxidoreductase (2-OR), pyruvate:ferredoxin oxidoreductase (PFOR) from Giardia duodenalis has been purified to apparent homogeneity. A second 2-OR with a preference for alpha-ketobutyrate as substrate was identified and was removed from PFOR containing fractions during purification. Only PFOR and the second 2-OR were identified in gels of crude Giardia extracts assayed for 2-OR activity. The native form of PFOR which is membrane associated, is a homodimer of 138 kDa subunits. Pyruvate is the preferred substrate: alpha-ketobutyrate and oxaloacetate, but not phenyl-pyruvate or alpha-ketoglutarate, are decarboxylated. PFOR from Giardia is more stable than PFOR from most other organisms and purified PFOR can be stored without deterioration at -70 degrees C. Purified PFOR donates electrons to Giardia ferredoxin (Fd I) with concomitant reduction of metronidazole. However, two other Giardia ferredoxins did not accept electrons from PFOR. Consistent with the involvement of PFOR in metronidazole activation, the activity of pyruvate dependent 2-OR activity was decreased in all metronidazole-resistant lines tested but not in furazolidone-resistant lines. The presence of three different ferredoxins and two 2-ORs in Giardia suggests that a number of different electron transport pathways operate in this organism providing unusual metabolic flexibility for a eukaryote.

Animals↗
PubMed1982

Two different species of messenger RNAs specify synthesis of M1 and M2 pyruvate kinase subunits.

A study was performed to determine whether M1 and M2 pyruvate kinases were synthesized under the direction of one or two messenger RNAs. We compared M1 and M2 pyruvate kinases purified from fresh tissues with those neosynthesized under the direction of messenger RNAs from tissues synthesizing either M1 or M2. RNA was isolated from rat muscle, lung, spleen and kidney by ethanol precipitation in 7 M guanidium chloride, translated in rabbit reticulocyte system and newly-synthesized pyruvate kinase subunits were purified by microimmunoaffinity chromatography. Pyruvate kinase from fresh muscle and spleen was purified in one step by a similar process. Muscle and spleen RNA directed the synthesis of M subunits with molecular weights of approx. 61000 and 62000, respectively, the same as those of the corresponding fresh tissue monomers. In addition, peptide maps obtained by partial digestion of neosynthesized M1 and M2 with V8 protease from Staphylococcus aureus confirmed that these polypeptides were clearly different.

Animals↗
PubMed1982

Affinity labeling of rabbit muscle pyruvate kinase with dialdehyde-ADP.

Periodate-oxidized ADP (dialdehyde-ADP) inactivates rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) and combines irreversibly to the enzyme. This inactivation is first-order with respect to dialdehyde-ADP and follows saturation kinetics, indicating that the enzyme first forms a reversible complex with the inactivator. Low Mg2+ concentrations stimulate the rate of inactivation, while higher concentrations have a protective effect. ADP and ATP, especially in the presence of Mg2+, protect very strongly against inactivation, while phosphoenolpyruvate and pyruvate are less effective. Dialdehyde-ADP is not a substrate, but acts as competitive inhibitor of ADP, with a KI of 4.5 mM. The analog has somewhat lower affinity to the enzyme than Mg-ADP, which has a Kd of 1.2 mM. Based on kinetic data, it is shown that one molecule of reagent must combine per enzyme active site in order to inactivate the enzyme. Incorporation of [14-C]dialdehyde-ADP to the enzyme and treatment of the data by the Tsou plot shows that 6-7 residues per subunit react with the modifier, two of them being essential for activity. From the evidence presented it is concluded: (1) dialdehyde-ADP behaves as an affinity label of rabbit muscle pyruvate kinase; (2) the inactivator binds probably to lysine residues at or near the active site, forming morpholine-like structures, and (3) the enzyme possesses two modifiable groups essential for activity, the reaction of one of them being sufficient to cause total loss in activity.

Adenosine Diphosphate↗
PubMed1985

The effect of pH on the interaction of substrates and effector to yeast and rabbit muscle pyruvate kinase.

The interaction of fructose 1,6-bisphosphate, phosphoenolpyruvate and ADP with pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from yeast and rabbit muscle has been studied as a function of pH utilizing the quenching of protein fluorescence at 330 nm by these ligands. Both the muscle and the yeast pyruvate kinase interact with either ADP or phosphoenolpyruvate with similar affinity, indicating that the substrate-binding sites for these two isozymes are similar. The major difference between the yeast and muscle isozymes is their affinity with fructose 1,6-bisphosphate. Fructose 1,6-bisphosphate interacts with the yeast isozyme in orders of magnitude more strongly than with the muscle isozyme. Moreover, the affinity of fructose 1,6-bisphosphate to the yeast isozyme is strongly pH-dependent, while the interaction of fructose 1,6-bisphosphate with the muscle isozyme is independent of pH. The data indicate that yeast pyruvate kinase undergoes a conformational change as the pH is increased from 6.0 to 8.5.

Adenosine Diphosphate↗
PubMed1989

Interaction between catalytic and regulatory sites of the pyruvate dehydrogenase from Escherichia coli studied by the ESR technique.

The accessibility of sulfhydryl groups at the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was reinvestigated. Hydrophobic interactions appear to control the reactivity of an essential cysteine residue at the active site with thiol reagents. This explains why the essential cysteine residue reacts only with thiol reagents of minor polarity, like p-hydroxymercuribenzoate or phenylmercuric nitrate, but not with Ellman's reagent or jodoacetamide. The pyruvate dehydrogenase component was modified with a nitroxide derivative of p-hydroxymercuribenzoate. The ESR spectrum of the spin-labelled enzyme changed dramatically upon addition of the cofactors thiamine diphosphate and Mg2+. Obviously spin-spin interaction occurs under these conditions caused by a transition of an inactive to an active state of the enzyme. The same conformational change is observed when the allosteric activator AMP instead of the cofactors was bound to the enzyme. The implications of these results for the allosteric regulation of the pyruvate dehydrogenase complex are discussed.

Dithionitrobenzoic Acid↗