Phosphorylation is switch of L-type pyruvate kinase allostery

2010 ◽  
Vol 5 (2) ◽  
pp. 135-142 ◽  
Author(s):  
Ilona Faustova ◽  
Aleksei Kuznetsov ◽  
Erkki Juronen ◽  
Mart Loog ◽  
Jaak Järv
Keyword(s):  
Pyruvate Kinase ◽  
Hill Coefficient ◽  
Stoichiometric Ratio ◽  
Allosteric Enzyme ◽  
E Coli ◽  
Glycolysis Regulation ◽  
Regulatory Phosphorylation ◽  
Pyruvate Kinase Isoenzymes ◽  
Dependent Protein ◽  
The Hill

AbstractAmong four pyruvate kinase isoenzymes, M1, M2, R and L, only M1 is considered as a nonallosteric enzyme. However, here we show that the non-phosphorylated L-type pyruvate kinase (L-PK) is also a non-allosteric enzyme with respect to its substrate phosphoenolpyruvate (PEP). The allosteric catalytic properties of L-PK are switched on through phosphorylation by cAMP-dependent protein kinase. The non-phosphorylated enzyme was produced by expressing the rat L-PK in E. coli, as the bacterium does not have mammalian-type protein kinases. The resulting tetrameric protein was phosphorylated with a stoichiometric ratio of one mole of phosphate per one L-PK monomer. Activity of the phosphorylated enzyme was allosterically regulated by PEP with the Hill coefficient n=2.5. It was observed that allostery was engaged by phosphorylation of the first subunit in the tetrameric enzyme, while further phosphorylation only modulated this effect. The discovered switching between non-allosteric and allosteric forms of L-PK and the possibility of modulating the allostery by phosphorylation are important for understanding of the interrelationship between allostery and the regulatory phosphorylation in general, and may have implication for further analysis of glycolysis regulation in the liver.

scholarly journals Kinetic studies on the regulation of rabbit liver pyruvate kinase

1973 ◽  
Vol 131 (2) ◽  
pp. 287-301 ◽  
Author(s):  
M. G. Irving ◽  
J. F. Williams
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Rabbit Liver ◽  
Hill Coefficient ◽  
Saturation Curve ◽  
Low Concentrations ◽  
Ammonium Sulphate Fractionation ◽  
The Hill

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K+ and optimum activity was recorded with 30mm-K+, 4mm-MgADP-, with a MgADP-/ADP2- ratio of 50:1, but inhibition occurred with K+ concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg2+ was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (nH+2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent Km for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis–Menten response was obtained when phosphoenolpyruvate was the variable substrate (Km+0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg2+.

Regulation of pyruvate kinase by 6-phosphogluconate in isolated hepatocytes

1981 ◽  
Vol 240 (3) ◽  
pp. E279-E285
Author(s):  
S. B. Smith ◽  
R. A. Freedland
Keyword(s):  
Pyruvate Kinase ◽  
Parenchymal Cell ◽  
Isolated Hepatocytes ◽  
Hill Coefficient ◽  
Liver Parenchymal Cell ◽  
Liver Parenchymal ◽  
Highly Correlated ◽  
Parenchymal Cells ◽  
The Hill ◽  
Isolated Liver

Isolated liver parenchymal cells from rats fed a 65% sucrose diet for 14 days were incubated in the presence and absence of 10(-6) M glucagon. The pyruvate kinase obtained from homogenates of the glucagon-treated cells displayed and increased Ks 0.5 for phosphoenolpyruvate (P-enolpyruvate), as well as an increased Ka 0.5 for 6-phosphogluconate (6-P-gluconate), compared to pyruvate kinase from untreated cells. Additionally, glucagon treatment decreased the maximal stimulation of pyruvate kinase by 6-P-gluconate by approximately two-thirds and decreased the Hill coefficient value of pyruvate kinase for 6-P-gluconate from 1.76 to 1.56. 6-Aminonicotinamide, an inhibitor of 6-P-gluconate dehydrogenase, increased 6-P-gluconate levels in isolated liver parenchymal cells three- to sevenfold, depending on the substrates present. The flux of P-enolpyruvate through pyruvate kinase was increased from 18 to 40% in these preparations and was highly correlated with the increase in 6-P-gluconate levels. The results suggest that 6-P-gluconate could regulate pyruvate kinase activity in the intact liver parenchymal cell. Furthermore, the activator would be of greatest importance in the lipogenic animal.

Oxygen transport, tissue glycogen stores, and tissue pyruvate kinase activity in muskrats

1985 ◽  
Vol 63 (6) ◽  
pp. 1440-1444 ◽  
Author(s):  
Gregory K. Snyder ◽  
Edward L. Binkley
Keyword(s):  
Pyruvate Kinase ◽  
Gastrocnemius Muscle ◽  
Saturation Pressure ◽  
Anaerobic Capacity ◽  
Hemoglobin Concentration ◽  
Hill Coefficient ◽  
Terrestrial Mammals ◽  
Oxygen Stores ◽  
Glycogen Stores ◽  
The Hill

Oxygen stores, respiratory properties of blood, tissue glycogen concentrations, and the rate-limiting enzyme for glycolysis, pyruvate kinase, were examined in muskrats (Ondatra zibethicus). Of the potential oxygen stores, lung volume (19.6 ± 0.68 mL/kg), and blood hemoglobin concentration (13.0 ± 0.34 g Hb/100 mL blood) were typical of terrestrial mammals, while concentrations of myoglobin in heart (7.4 ± 0.1 mg Mb/g tissue) and gastrocnemius muscle (13.3 ± 0.5 mg Mb/g tissue) were significantly higher than values from the same tissues in the laboratory rat. Blood P50 (oxygen half-saturation pressure) at pH 7.4, 24.4 ± 1.36 Torr (1 Torr = 133.322 Pa), and Bohr effect, −0.64 ± 0.04, also were significantly different, while the Hill coefficient and buffering capacity were comparable for both species. Of our measures of anaerobic capacity, glycogen concentrations and pyruvate kinase activities in heart, brain, and gastrocnemius muscle of O. zibethicus were all comparable to values obtained for terrestrial mammals. We conclude that muskrats tolerate submersion by adaptations associated with aerobic metabolism, although a review of the literature shows that these adaptations are fully developed only in animals freshly captured during winter.

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

1977 ◽  
Vol 55 (6) ◽  
pp. 618-624 ◽  
Author(s):  
Robin C. McKellar ◽  
Donn J. Kushner
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Kinase ◽  
Hydrophobic Interactions ◽  
Glycolytic Enzyme ◽  
Molecular Probe ◽  
Inhibitory Effects ◽  
E Coli ◽  
The Hill ◽  
Escherichia Coli B ◽  
Inhibition Curve

Proflavine (PF) inhibited glucose use in sensitive but not resistant Escherichia coli B. Glucose transport (as measured by α-methylglucoside accumulation) was only partly inhibited by PF concentrations that completely blocked glucose use. Fructose 1,6-diphosphate-(FDP)-regulated pyruvate kinase (PK I) (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 K1/2 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, fructose 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 than PK I to the inhibitory effects of PF. A role for PF as a molecular probe for PK I has been proposed.

scholarly journals Factors affecting the activity of pyruvate kinase of Acetobacter xylinum

1969 ◽  
Vol 112 (5) ◽  
pp. 631-636 ◽  
Author(s):  
Moshe Benziman
Keyword(s):  
Pyruvate Kinase ◽  
Tricarboxylic Acid Cycle ◽  
Relative Activity ◽  
Acetobacter Xylinum ◽  
Hill Coefficient ◽  
Factors Affecting ◽  
Phosphofructokinase Activity ◽  
Ammonium Sulphate Fractionation ◽  
Relationship Of ◽  
The Hill

1. Extracts of Acetobacter xylinum were found to contain the glycolytic enzymes involved in the conversion of triose phosphate into pyruvate. Pyruvate kinase had the lowest relative activity. Phosphofructokinase activity was not detected in the extracts. 2. Only slight differences in the activity of pyruvate kinase were observed between cells grown on glucose and those grown on intermediates of the tricarboxylic acid cycle. 3. Pyruvate kinase, partially purified from ultrasonic extracts by ammonium sulphate fractionation, required Mg2+ ions for activity. It was not activated by K+ or NH4+ ions. 4. The plots representing the relationship between initial velocity and phosphoenolpyruvate concentration were sigmoidal, suggesting a co-operative effect for phosphoenolpyruvate. The Hill coefficient (n) for phosphoenolpyruvate was 2. The rate of the reaction changed with increasing ADP concentrations according to normal Michaelis–Menten kinetics. 5. The enzyme was inhibited by ATP (Ki0·9×10−3m). The inhibition was competitive with regard to ADP but not with regard to phosphoenolpyruvate. It was not relieved by excess of Mg2+ ions. 6. The possible relationship of the properties of pyruvate kinase to regulatory mechanisms for controlling gluconeogenesis and carbohydrate oxidation in A. xylinum is discussed.

scholarly journals Occurrence of paradoxical or sustained control by an enzyme when overexpressed: necessary conditions and experimental evidence with regard to hepatic glucokinase

2001 ◽  
Vol 355 (3) ◽  
pp. 787-793 ◽  
Author(s):  
Pedro DE ATAURI ◽  
Luis ACERENZA ◽  
Boris N. KHOLODENKO ◽  
Núria DE LA IGLESIA ◽  
Joan J. GUINOVART ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Gain Control ◽  
Hill Coefficient ◽  
Necessary Condition ◽  
Control Coefficient ◽  
Allosteric Enzyme ◽  
Flux Control ◽  
Sigmoidal Kinetics ◽  
The Hill ◽  
Pathway Flux

It is widely assumed that the control coefficient of an enzyme on pathway flux decreases as the concentration of enzyme increases. However, it has been shown [Kholodenko and Brown (1996) Biochem. J. 314, 753–760] that enzymes with sigmoidal kinetics can maintain or even gain control with an increase in enzyme activity or concentration. This has been described as ‘paradoxical control’. Here we formulate the general requirements for allosteric enzyme kinetics to display this behaviour. We show that a necessary condition is that the Hill coefficient of the enzyme should increase with an increase in substrate concentration or decrease with an increase in product concentration. We also describe the necessary and sufficient requirements for the occurrence of paradoxical control in terms of the flux control coefficients and the derivatives of the elasticities. The derived expression shows that the higher the control coefficient of an allosteric enzyme, the more likely it is that the pathway will display this behaviour. Control of pathway flux is generally shared between a large number of enzymes and therefore the likelihood of observing sustained or increased control is low, even if the kinetic parameters are in the most favourable range to generate the phenomenon. We show that hepatic glucokinase, which has a very high flux control coefficient and displays sigmoidal behaviour within the hepatocyte in situ as a result of interaction with a regulatory protein, displays sustained or increased control over an extended range of enzyme concentrations when the regulatory protein is overexpressed.

On the Measurement of Cooperativity and the Physico-Chemical Meaning of the Hill Coefficient

2019 ◽  
Vol 20 (9) ◽  
pp. 861-872 ◽  
Author(s):  
Andrea Bellelli ◽  
Emanuele Caglioti
Keyword(s):  
Ligand Binding ◽  
Hormone Receptors ◽  
Fundamental Property ◽  
Statistical Correlation ◽  
Hill Coefficient ◽  
Biological Macromolecules ◽  
Physico Chemical ◽  
Physiological Relevance ◽  
Minimum Estimate ◽  
The Hill

Cooperative ligand binding is a fundamental property of many biological macromolecules, notably transport proteins, hormone receptors, and enzymes. Positive homotropic cooperativity, the form of cooperativity that has greatest physiological relevance, causes the ligand affinity to increase as ligation proceeds, thus increasing the steepness of the ligand-binding isotherm. The measurement of the extent of cooperativity has proven difficult, and the most commonly employed marker of cooperativity, the Hill coefficient, originates from a structural hypothesis that has long been disproved. However, a wealth of relevant biochemical data has been interpreted using the Hill coefficient and is being used in studies on evolution and comparative physiology. Even a cursory analysis of the pertinent literature shows that several authors tried to derive more sound biochemical information from the Hill coefficient, often unaware of each other. As a result, a perplexing array of equations interpreting the Hill coefficient is available in the literature, each responding to specific simplifications or assumptions. In this work, we summarize and try to order these attempts, and demonstrate that the Hill coefficient (i) provides a minimum estimate of the free energy of interaction, the other parameter used to measure cooperativity, and (ii) bears a robust statistical correlation to the population of incompletely saturated ligation intermediates. Our aim is to critically evaluate the different analyses that have been advanced to provide a physical meaning to the Hill coefficient, and possibly to select the most reliable ones to be used in comparative studies that may make use of the extensive but elusive information available in the literature.

Effect of adrenomedullin on the production of endothelin-1 and on its vasoconstrictor action in resistance arteries: evidence for a receptor-specific functional interaction in patients with heart failure

2001 ◽  
Vol 101 (1) ◽  
pp. 45-51 ◽  
Author(s):  
Chris HILLIER ◽  
Mark C. PETRIE ◽  
Michael P. LOVE ◽  
Fiona JOHNSTON ◽  
Margaret R. MACLEAN ◽  
...  
Keyword(s):  
Heart Failure ◽  
Hill Coefficient ◽  
Receptor Interaction ◽  
Resistance Arteries ◽  
Functional Interaction ◽  
High Potassium ◽  
Endothelin 1 ◽  
Patients With Heart Failure ◽  
Small Resistance ◽  
The Hill

Endothelin-1 (ET-1) and adrenomedullin (ADM) are both produced in the arterial wall, but have opposing biological actions. Evidence from experimental animals suggests a functional interaction between ET-1 and ADM. We have tested this in humans. Small resistance arteries were obtained from gluteal biopsies taken from patients with chronic heart failure (CHF) due to coronary heart disease (CHD), or with CHD and preserved ventricular function. The contractile responses to big ET-1 and to ET-1 in both sets of vessels were studied in the absence (control) and presence of ADM at 20 pmol/l (low ADM) or 200 pmol/l (high ADM), using wire myography. ADM did not affect the conversion of big ET-1 into ET-1 in vessels from patients with either CHD or CHF. Low ADM did not alter the contractile response to ET-1 in vessels from patients with CHF. Low ADM was not tested in vessels from patients with CHD, but high ADM did not affect this response in arteries from these patients. High ADM did, however, significantly reduce the vasoconstrictor effect of ET-1 in vessels from patients with CHF. The maximum response, as a percentage of the response to high potassium, was 199% (S.E.M. 25%) in the control experiments (n = 14), 205% (27%) in the low-ADM (n = 7) studies and 150% (17%) in the high-ADM (n = 6) experiments (P < 0.001). Furthermore, the Hill coefficient increased from 0.57±0.05 in the absence of ADM to 1.16±0.15 in the high-ADM experiments, indicating that ADM at 200 pmol/l specifically antagonized one receptor type in vessels from patients with CHF. We conclude that there is a one-site receptor interaction between ADM and ET-1 that is specific for vessels from patients with CHF. This functional interaction between ADM and ET-1 in resistance arteries may be of pathophysiological importance in CHF.

scholarly journals Activation and Two Modes of Blockade by Strontium of Ca2+-activated K+ Channels in Goldfish Saccular Hair Cells

1998 ◽  
Vol 111 (2) ◽  
pp. 363-379 ◽  
Author(s):  
Izumi Sugihara
Keyword(s):  
Dissociation Constant ◽  
Time Constant ◽  
Hair Cells ◽  
Single Channel ◽  
Hill Coefficient ◽  
K Channels ◽  
Voltage Dependent ◽  
Time Courses ◽  
The Hill ◽  
Inside Out

Effects of internal Sr2+ on the activity of large-conductance Ca2+-activated K+ channels were studied in inside-out membrane patches from goldfish saccular hair cells. Sr2+ was approximately one-fourth as potent as Ca2+ in activating these channels. Although the Hill coefficient for Sr2+ was smaller than that for Ca2+, maximum open-state probability, voltage dependence, steady state gating kinetics, and time courses of activation and deactivation of the channel were very similar under the presence of equipotent concentrations of Ca2+ and Sr2+. This suggests that voltage-dependent activation is partially independent of the ligand. Internal Sr2+ at higher concentrations (&gt;100 μM) produced fast and slow blockade both concentration and voltage dependently. The reduction in single-channel amplitude (fast blockade) could be fitted with a modified Woodhull equation that incorporated the Hill coefficient. The dissociation constant at 0 mV, the Hill coefficient, and zd (a product of the charge of the blocking ion and the fraction of the voltage difference at the binding site from the inside) in this equation were 58–209 mM, 0.69–0.75, 0.45–0.51, respectively (n = 4). Long shut events (slow blockade) produced by Sr2+ lasted ∼10–200 ms and could be fitted with single-exponential curves (time constant, τl−s) in shut-time histograms. Durations of burst events, periods intercalated by long shut events, could also be fitted with single exponentials (time constant, τb). A significant decrease in τb and no large changes in τl−s were observed with increased Sr2+ concentration and voltage. These findings on slow blockade could be approximated by a model in which single Sr2+ ions bind to a blocking site within the channel pore beyond the energy barrier from the inside, as proposed for Ba2+ blockade. The dissociation constant at 0 mV and zd in the Woodhull equation for this model were 36–150 mM and 1–1.8, respectively (n = 3).

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