Regulation of the pyruvate kinase from Alcaligenes eutrophus H 16 in vitro and in vivo

The biosynthesis of the enzyme pyruvate kinase (E.C. 2.7.1.40) of Alcaligenes eutrophus (Hydrogenomonas eutropha) H 16 was influenced by the carbon and energy source. After growth on gluconate the specific enzyme activity was high while acetate grown cells exhibited lower activities (340 and 55 μmoles/min·g protein, respectively). The pyruvate kinase from autotrophically grown cells was purified 110-fold. The enzyme was characterized by homotropic cooperative interactions with the substrate phosphoenolpyruvate, the activators AMP, ribose-5-phosphate, glucose-6-phosphate and the inhibitor ortho-phosphate. In addition to phosphate ATP caused inhibition but in this case non-sigmoidal kinetics was obtained. The half maximal substrate saturation constant S_0.5 for phosphoenolpyruvate in the absence of any effectors was 0.12 mM, in the presence of 1 mM ribose-5-phosphate 0.07 mM, and with 9 mM phosphate 0.67 mM. The corresponding Hill values were 0.96, 1.1 and 2.75. The ADP saturation curve was hyperbolic even in the presence of the effectors, the K _m value was 0.14 mM ADP. When the known intracellular metabolite concentrations in A. eutrophus H 16 were compared with the regulatory sensitivity of the enzyme, it appeared that under the conditions in vivo the inhibition by ATP was more important than the regulation by the allosteric effectors.     

Control of pyruvate kinase activity during glycolysis and gluconeogenesis in Propionibacterium shermanii

The concentrations of glycolytic intermediates and ATP and the activities of certain glycolytic and gluconeogenic enzymes were determined in Propionibacterium shermanii cultures grown on a fully defined medium with glucose, glycerol or lactate as energy source. On all three energy sources, enzyme activities were similar and pyruvate kinase was considerably more active than the gluconeogenic enzyme pyruvate, orthophosphate dikinase, indicating the need for regulation of pyruvate kinase activity. The intracellular concentration of glucose 6-phosphate, a specific activator of pyruvate kinase in this organism, changed markedly according to both the nature and the concentration of the growth substrate: the concentration (7-10 mM) during growth with excess glucose or glycerol was higher than that (1-2 mM) during growth with lactate or at growth-limiting concentrations of glycerol or glucose. Other glycolytic intermediates, apart from pyruvate, were present at concentrations below 2 mM. Glucose 6-phosphate overcame inhibition of pyruvate kinase activity by ATP and inorganic phosphate. With 1 mM-ATP and more than 10 mM inorganic phosphate, a change in glucose 6-phosphate concentration from 1-2 mM was sufficient to switch pyruvate kinase from a strongly inhibited to a fully active state. The results provide a plausible mechanism for the regulation of glycolysis and gluconeogenesis in P. shermanii.     

[3H]proline incorporation and hydroxyproline concentration in articular cartilage during the development of osteoarthritis caused by immobilization. A study in vivo with rabbits.

1. Activation of glucose 6-phosphate is one of the unique properties of pyruvate kinase from Mycobacterium smegmatis. 2. Pyruvate kinase, partially purified from ultrasonic extracts of the mycobacteria by (NH4)2SO4 fractionation, exhibited sigmoidal kinetics at various concentrations of phosphoenolpyruvate, with a high degree of co-operativity (Hill coefficient, h = 3.7) and S0.5 value of 1.0 mM. 3. In the presence of glucose 6-phosphate, the degree of co-operativity shown by the phosphoenolpyruvate saturation curve was decreased to h = 2.33 and the S0.5 value was lowered to 0.47 mM. 4. The enzyme was activated by AMP and ribose 5-phosphate also, but the activation constant was lowest with glucose 6-phosphate (0.24 mM). 5. The enzyme was strongly inhibited by ATP at all phosphoenolpyruvate concentrations. The concentrations of ATP required to produce half-maximal inhibition of enzyme activity at non-saturating (0.2 mM) and saturating (2 mM) phosphoenolpyruvate concentrations were 1.1 mM and 3 mM respectively. 6. The inhibition of ATP was partially relieved by glucose 6-phosphate. 7. The enzyme exhibited Michaelis-Menten kinetics with ADP as the variable substrate, with an apparent Km of 0.66 mM. 8. The enzyme required Mg2+ or Mn2+ ions for activity. It was not activated by univalent cations. 9. The kinetic data indicate that under physiological conditions glucose 6-phosphate probably plays a significant role in the regulation of pyruvate kinase activity.     

Regulation of pyruvate kinase from Propionibacterium shermanii

Pyruvate kinase from Propionibacterium shermanii was shown to be activated by glucose-6-phosphate (G-6-P) at non-saturating phosphoenol pyruvate (PEP) concentrations but other glycolytic and hexose monophosphate pathway intermediates and AMP were without effect. Half-maximal activation was obtained at 1 mM G-6-P. The presence of G-6-P decreased both the PEP_0.5V and ADP_0.5V values and the slope of the Hill plots for both substrates. The enzyme was strongly inhibited by ATP and inorganic phosphate (P_i) at all PEP concentrations. At non-saturating (0.5 mM) PEP, half-maximal inhibition was obtained at 1.8 mM ATP or 1.4 mM P_i. The inhibition by both P_i and ATP was largely overcome by 4 mM G-6-P. The specific activity of pyruvate kinase was considerably higher in lactate-, glucose- and glycerol-grown cultures than that of the enzyme catalysing the reverse reaction, pyruvate, phosphate dikinase. It is suggested that the activity of pyruvate kinase in vivo is determined by the balance between activators and inhibitors such that it is inhibited during gluconeogenesis while, during glycolysis, the inhibition is relieved by G-6-P.Abbreviations PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate - P_i inorganic phosphate     

Glucose requirement for postischemic recovery of perfused working heart

The quantitative importance of glycolysis in cardiomyocyte reenergization and contractile recovery was examined in postischemic, preload-controlled, isolated working guinea pig hearts. A 25-min global but low-flow ischemia with concurrent norepinephrine infusion to exhaust cellular glycogen stores was followed by a 15-min reperfusion. With 5 mM pyruvate as sole reperfusion substrate, severe contractile failure developed despite normal sarcolemmal pyruvate transport rate and high intracellular pyruvate concentrations near 2 mM. Reperfusion dysfunction was characterized by a low cytosolic phosphorylation potential [( ATP]/[( ADP][Pi]) due to accumulations of inorganic phosphate (Pi) and lactate. In contrast, with 5 mM glucose plus pyruvate as substrates, but not with glucose as sole substrate, reperfusion phosphorylation potential and function recovered to near normal. During the critical ischemia-reperfusion transition at 30 s reperfusion the cytosolic creatine kinase appeared displaced from equilibrium, regardless of the substrate supply. When under these conditions glucose and pyruvate were coinfused, glycolytic flux was near maximum, the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction was enhanced, accumulation of Pi was attenuated, ATP content was slightly increased, and adenosine release was low. Thus, glucose prevented deterioration of the phosphorylation potential to levels incompatible with reperfusion recovery. Immediate energetic support due to maximum glycolytic ATP production and enhancement of the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction appeared to act in concert to prevent detrimental collapse of [ATP]/[( ADP][Pi]) during creatine kinase dysfunction in the ischemia-reperfusion transition. Dichloroacetate (2 mM) plus glucose stimulated glycolysis but failed fully to reenergize the reperfused heart; conversely, 10 mM 2-deoxyglucose plus pyruvate inhibited glycolysis and produced virtually instantaneous de-energization during reperfusion. The following conclusions were reached. (1) A functional glycolysis is required to prevent energetic and contractile collapse of the low-flow ischemic or reperfused heart (2). Glucose stabilization of energetics in pyruvate-perfused hearts is due in part to intensification of glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase activity. (3) 2-Deoxyglucose depletes the glyceraldehyde-3-phosphate pool and effects intracellular phosphate fixation in the form of 2-deoxyglucose 6-phosphate, but the cytosolic phosphorylation potential is not increased and reperfusion failure occurs instantly. (4) Consistent correlations exist between cytosolic ATP phosphorylation potential and reperfusion contractile function. The findings depict glycolysis as a highly adaptive emergency mechanism which can prevent deleterious myocyte deenergization during forced ischemia-reperfusion transitions in presence of excess oxidative substrate.     

PURIFICATION AND CHARACTERIZATION OF PYRUVATE KINASE FROM THE GREEN ALGA CHLAMYDOMONAS REINHARDTII1

A single form of pyruvate kinase was isolated from the green alga Chlamydomonas reinhardtii Dang. (Chlorophyta) and partially purified over twentyfold, yielding a final specific activity of 2.68 μmol pyruvate produced-min^-1.mg^-1 protein. Studies of its physical characteristics reveal that the pyruvate kinase is heat stable, is partially inactivated by sulfhydryl reagent N-ethylmaleimide, and has a pH optimum at 6.8 and a native molecular mass of 224 kDa. Immunological precipitation and western blotting, using antibodies raised against Selenastrum minutum Naeg. (Chlorophyta) cytosolic pyruvate kinase, reveal that C. reinhardtii pyruvate kinase possesses a subunit molecular mass of 57 kDa, indicating a homo-tetrameric structure. This enzyme exhibits an absolute requirement for a divalent cation that can be fulfilled, by Mg²⁺. The monovalent cation K⁺ acts as a strong activator. The K_m values for phosphoenolpyruvate and adenosine diphosphate (ADP) are 0.16 mM and 0.18 mM, respectively. The enzyme is capable of using other nucleotides with V_max for UDP, GDP, IDP, and CDP of 70%, 55%, 53%, and 25% of that with ADP, respectively. Dihydroxyacetone phosphate, ribulose 1,5-bisphosphate, adenosine monophosphate (AMP), ribose-5-phosphate, and glyceraldehyde-3-phosphate are activators, whereas glutamate, orthophosphate, adenosine triphosphate (ATP), citrate, isocitrate, malate, oxalate, phosphoglycolate, and 2,3-diphosphoglycerate are potent inhibitors of this enzyme. Dihydroxyacetone phosphate can reverse the inhibition by glutamate and phosphate. These properties are discussed in light of pyruvate kinase regulation during anabolic and catabolic respiration. Substrate interaction and product inhibition studies indicate that ADP is the first substrate bound to the enzyme and pyruvate is the last product released (Ordered Bi Bi mechanism).     

The influence of inorganic phosphate and ATP on the kinetics of bovine heart muscle pyruvate kinase

Summary The mechanism of activation by inorganic phosphate and ATP of cardiac muscle pyruvate kinase was studied with the aid of steady-state kinetics. The enzyme was purified to homogeneity to a final specific activity of 400 units/ mg (phosphate buffer, pH 7.6, 25 °C). At pH 7.6 the enzyme displays Michaelis-Menten kinetics with respect to both its substrates, phosphoenolpyruvate and ADP. Substrate kinetic constants are: app.K_m(phosphoenolpyruvate) –0.04 mM, app.K_m(ADP) =0.22 mM. Under the conditions used in the standard assay the specific activity is greatly enhanced by inorganic phosphate (50 mM) or ATP (2.5 mM). Each of these modifiers, acting separately, increases the V_max without seriously affecting Michaelis constants and Hill coefficients. In the presence of both Pi and ATP, only a decrease in V_max was observed.The kinetics of activation by inorganic phosphate of pyruvate kinase was examined. Studying the effect of varying concentrations of Pi on the initial rate we obtained a hyperbolic saturation curve with the app. K_m(P_i) = 20 mM and V_max = 167 units/ mg. The evidence is presented that inorganic phosphate is a substrate for a side reaction catalyzed by cardiac pyruvate kinase. It is shown that in the presence of pyruvate, inorganic phosphate and ATP in the assay system, P_i is incorporated into acid-labile products of this reaction, inorganic pyrophosphate being one of them.These findings indicate the existence of an alternative reaction catalyzed by pyruvate kinase by which energy may be stored in the form of inorganic pyrophosphate.Abbreviations PEP phosphoenolpyruvate - Pi inorganic phosphate - TEA triethanolamine - EDTA ethylenediaminetetraacetate     

Studies on the kinetic effects of adenosine-3':5'-monophosphate-dependent phosphorylation of purified pig-liver pyruvate kinase type L.

The effect of cyclic-AMP-dependent phosphorylation on the activity of isolated pig liver pyruvate kinase was studied. It was found that the major kinetic effect of the phosphorylation was to reduce the affinity for the substrate phosphoenolpyruvate, K0.5 for this substrate increasing from 0.3 to 0.9 mM upon phosphorylation. The cooperative effect with phosphoenolpyruvate was enhanced, the Hill constant nH increasing concomitantly from 1.1 to 1.5. V was unaltered. The change in activity occurred in parallel with the phosphate incorporation, except during the initial part of the reaction, when inactivation was correspondingly slower. The affinity for the second substrate ADP was unchanged, with an apparent Km of 0.3 mM at saturating concentration of phosphoenolpyruvate. Likewise, the requirement for potassium was unaffected, whereas the phosphoenzyme required a higher concentration of magnesium ions for maximal activity, compared with the control enzyme. The inhibitory effect of the phosphorylation was counteracted by positive effectors, fructose 1,6-biphosphate in micromolar concentrations completely activated the phosphoenzyme, resulting in an enzyme with properties similar to the fructose 1,6-biphosphate-activated unphosphorylated enzyme, with K0.5 for phosphoenolpyruvate about 0.025 mM and with a Hill constant of 1.1. Hydrogen ions were also effective in activating the phosphoenzyme. Thus, when pH was lowered from 8 to 6.5 the inhibition due to phosphorylation was abolished. The phosphoenzyme was sensitive to further inhibition by negative effectors such as ATP and alanine. 2 mM ATP increased K0.5 for phosphoenolpyruvate to 1.5 mM and nH to 2.3. The corresponding values with alanine were 1.3 mM and 1.9. Phosphorylation is thought to be an additional mechanism of inhibition of the enzyme under gluconeogenetic conditions.     

Metabolic regulation of pyruvate kinase isolated from autotrophically and heterotrophically grown Paracoccus denitrificans

Pyruvate kinase (ATP: pyruvate phosphotransferase (EC 2.7.1.40) was partially purified from both autotrophically and heterotrophycally grown Paracoccus denitrificans. The organism grown under heterotrophic conditions contains four times more pyruvate kinase than under autotrophic conditions. The enzyme isolated from both sources exhibited sigmoidal kinetics for both phosphoenolpyruvate (PEP) and ADP. The apparent M _m for ADP and PEP in the autotrophic enzyme were 0.63 mM ADP and 0.25 mM PEP. The effect of several low molecular weight metabolites on the pyruvate kinase activity was investigated. Ribose-5-phosphate, glucose-6-phosphate and AMP stimulated the reaction at low ADP levels; this stimulation was brought about by an alteration in the apparent K _m for ADP. The pyruvate kinases differ in their response to adenine nucleotides, but both preparations seem to be under adenylate control. The results are discussed in relation to the role of pyruvate kinase as a regulatory enzyme in P. denitrificans grown under both autotrophic and heterotrophic conditions.Non-Common Abbreviations PEP phosphoenolpyruvate - R-5-P ribose-5-phosphate - G-6-P glucose-6-phosphate - F-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate     

Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase.

The control of pyruvate dehydrogenase activity by inactivation and activation was studied in intact mitochondria isolated from rabbit heart. Pyruvate dehydrogenase could be completely inactivated by incubating mitochondria with ATP, oligomycin, and NaF. This loss in dehydrogenase activity was correlated with the incorporation of 32P from [gamma-32P]ATP into mitochondrial protein(s) and with a decrease in the mitochondrial oxidation of pyruvate. ATP may be supplied exogenously, generated from endogenous ADP during oxidative phosphorylation, or formed from exogenous ADP in carbonyl cyanid p-trifluoromethoxyphenylhydrazone-uncoupled mitochondria. With coupled mitochondria the concentration of added ATP required to half-inactivate the dehydrogenase was 0.24 mM. With uncoupled mitochondria the apparent Km was decreased to 60 muM ATP. Inactivation of pyruvate dehydrogenase by exogenous ATP was sensitive to atractyloside, suggesting that pyruvate dehydrogenase kinase acts internally to the atractyloside-sensitive barrier. The divalent cation ionophore, A23187, enhanced the loss of dehydrogenase activity. Pyruvate dehydrogenase activity is regulated additionally by pyruvate, inorganic phosphate, and ADP. Pyruvate, in the presence of rotenone, strongly inhibited inactivation. This suggests that pyruvate facilitates its own oxidation and that increases in pyruvate dehydrogenase activity by substrate may provide a modulating influence on the utilization of pyruvate via the tricarboxylate cycle. Inorganic phosphate protected the dehydrogenase from inactivation by ATP. ADP added to the incubation mixture together with ATP inhibited the inactivation of pyruvate dehydrogenase. This protection may result from a direct action on pyruvate dehydrogenase kinase, as ADP competes with ATP, and an indirect action, in that ADP competes with ATP for the translocase. It is suggested that the intramitochondrial [ATP]:[ADP] ratio effects the kinase activity directly, whereas the cytosolic [ATP]:[ADP] ratio acts indirectly. Mg2+ enhances the rate of reactivation of the inactivated pyruvate dehydrogenase presumably by accelerating the rate of dephosphorylation of the enzyme. Maximal activation is obtained with the addition of 0.5 mM Mg2+..     

Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP

Pyruvate dehydrogenase kinase 2 (PDK2) activity is enhanced by the dihydrolipoyl acetyltransferase core (E2 60mer) that binds PDK2 and a large number of its pyruvate dehydrogenase (E1) substrate. With E2-activated PDK2, K(+) at approximately 90 mM and Cl(-) at approximately 60 mM decreased the K(m) of PDK2 for ATP and competitive K(i) for ADP by approximately 3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis +/- E2, E2 increased the K(m) of PDK2 for ATP by nearly 8-fold (from 5 to 39 microM), increased k(cat) by approximately 4-fold, and decreased the requirement for E1 by at least 400-fold. ATP binding, measured by a cold-trapping technique, occurred at two active sites with a K(d) of 5 microM, which equals the K(m) and K(d) of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had approximately 3 times more ADP than ATP bound at its active site, and the pyruvate analogue, dichloroacetate, led to 16-fold more ADP than ATP being bound (no added ADP). Pyruvate functioned as an uncompetitive inhibitor versus ATP, and inclusion of ADP transformed pyruvate inhibition to noncompetitive. At high pyruvate levels, pyruvate was a partial inhibitor but also induced substrate inhibition at high ATP levels. Our results indicate that, at physiological salt levels, ADP dissociation is a limiting step in E2-activated PDK2 catalysis, that PDK2.[ADP or ATP].pyruvate complexes form, and that PDK2.ATP.pyruvate.E1 reacts with PDK2.ADP.pyruvate accumulating.     

Activation of rat liver cytosolic phosphatidic acid phosphatase by nucleoside diphosphates

Phosphatidic acid phosphatase (EC 3.1.3.4) was purified 30-fold by ammonium sulfate fractionation and hydroxyapatite chromatography from the soluble fraction of rat liver. ADP was found to stimulate the enzyme activity with half-maximal stimulation at 0.2 mM. Similar effects were seen when ADP was replaced by GDP or CDP. In contrast, ATP inhibited the enzyme; half-maximal inhibition observed at 0.2 mM. Again, the degree of inhibition did not differ when GTP or CTP replaced ATP. Thus, the structure of the base part of the nucleotide was not critical for mediating these effects. The positions of the phosphate groups in the nucleotide structure were however found to be of importance for the enzyme activity. Variations in the structure of the phosphate ester bound at the 5'-position had a pronounced effect on phosphatidic acid phosphatase activity. The effect of nucleotides depended on pH, and the inhibition by ATP was more pronounced at pH levels lower than 7.0, whereas the stimulatory effect of ADP was virtually the same from pH 6.0 to pH 8.0. The enzyme showed substrate saturation kinetics with respect to phosphatidic acid, with an apparent Km of 0.7 mM. Km increased in the presence of ATP, whereas both apparent Vmax and Km increased in the presence of ADP, suggesting different mechanisms for the action of the two types of nucleotides. The results indicated that physiological levels of nucleotides with a diphosphate or a triphosphate ester bound at the 5'-position of the ribose moiety influenced the activity of phosphatidic acid phosphatase. The possibility is discussed that these effects might be of importance for the regulation of triacylglycerol biosynthesis.     

Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from Plasmodium falciparum

The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K_m of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1 mM pyridoxal-5′-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K_i of 0.8 mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.     

Purification and properties of pyruvate kinase from Streptococcus mutans.

Pyruvate kinase (EC 2.7.1.40) from Streptococcus mutans strain JC2 was purified, giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was 180,000 to 190,000, and the enzyme was considered to consist of four identical subunits. This enzyme was completely dependent on glucose 6-phosphate for activity, and the saturation curve for activation by glucose 6-phosphate was sigmoidal. In the presence of 0.5 mM glucose 6-phosphate, the saturation curves for the substrates phosphoenolpyruvate and ADP were hyperbolic, and the Km values were 0.22 and 0.39 mM, respectively. GDP, IDP, and UDP could replace ADP, and the Km for GDP (0.026 mM) was 0.067 of that for ADP. The enzyme required not only divalent cations, Mg2+ or Mn2+, but also monovalent cations, K+ or NH4+, for activity, and it was strongly inhibited by Pi. When the concentration of Pi was increased, the half-saturating concentration and Hill coefficient for glucose 6-phosphate increased. However, the enzyme was immediately inactivated in a solution without Pi. The intracellular concentration of glucose 6-phosphate, in cooperation with that of Pi, may regulate pyruvate kinase activity in S. mutans.     

The internal pH and membrane potential of the insulin-secretory granule

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.     

Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from Plasmodium falciparum

The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K(m) of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1mM pyridoxal-5'-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K(i) of 0.8mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.     

Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite

After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F₁ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfiteor nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - P_i inorganic orthophosphate Dedicated to Prof. Dr. Hans Grisebach on the occasion of his sixtieth birthday     

Chloroplast phosphofructokinase in the green alga, Dunaliella marina: partial purification and kinetic and regulatory properties

The aim of this work was to study the pathway(s) of sugar phosphate metabolism in chloroplasts of the unicellular green alga, Dunaliella marina (Volvocales). Phosphofructokinase, detectable in crude cell extracts, copurifled with intact chloroplasts on sucrose density gradients. In isolated chloroplasts, phosphofructokinase activity displayed latency to the same degree as chloroplast marker enzymes. From the quantitative distribution of enzyme activities in fractionated cells, it is concluded that there is an exclusive localization of phosphofructokinase in chloroplasts. In addition, no separation into multiple forms could be achieved. For the study of regulatory properties, chloroplast phosphofructokinase was partially purified by ammonium sulfate fractionation followed by DEAE-cellulose chromatography. The pH optimum of the enzyme activity was 7.0 and was not altered with varying concentrations of substrates or low-molecular-weight effectors. Fructose 6-phosphate showed a sigmoidal saturation curve whose shape was further changed with varying protein concentrations of the preparation. The second substrate, ATP, gave a hyperbolic saturation curve with a Michaelis constant of 60 μm. At a Mg²⁺ concentration of 2.5 mm, ATP concentrations exceeding 1 mm inhibited the enzyme in a positive cooperative manner. The same type of inhibition was observed with other phosphorylated intermediates of carbon metabolism, the most efficient being phosphoenolpyruvate, glycolate 2-phosphate, glycerate 3-phosphate, and glycerate 2-phosphate. Inorganic phosphate was the only activator found for phosphofructokinase. With nonsaturating fructose 6-phosphate concentrations, P_i activated in a positive cooperative fashion, while no activation occurred with saturating fructose 6-phosphate concentrations. In the presence of either an activator or an inhibitor, the sigmoidal shape of the fructose 6-phosphate saturation curve was altered. Most notably, the activator P_i could relieve the inhibitory action of ATP, phosphoenolpyruvate, glycerate 3-phosphate, glycerate 2-phosphate, and glycolate 2-phosphate. Based on these experimental findings, the regulatory properties of D. marina chloroplast phosphofructokinase are discussed with respect to its playing a key role in the regulation of chloroplast starch metabolism during a light/dark transition. All available evidence is compatible with the interpretation that phosphofructokinase is active only in the dark thus channeling starch degradation products into glycolysis.     

Purification and characterizaton of plastidic pyruvate kinase from developing seeds of Brassica campestris L.

Plastidic pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) was purified to near homogeneity as judged by native PAGE with about 4% recovery from developing seeds of Brassica campestris using (NH4)2SO4 fractionation, DEAE-cellulose chromatography, gel filtration through Sepharose-CL-6B and affinity chromatography through reactive blue Sepharose-CL-6B. The purified enzyme having molecular mass of about 266 kDa was quite stable and showed a broad pH optimum between pH 6.8-7.8. Typical Michaelis-Menten kinetics was obtained for both the substrates with K(m) values of 0.13 and 0.14 mM for PEP and ADP, respectively. The enzyme could also utilize CDP, GDP or UDP as alternative nucleotide to ADP, but with lower Vmax and higher K(m). The enzyme had an absolute requirement for a divalent and a monovalent cation for activity and was inhibited by oxalate, fumarate, citrate, isocitrate and ATP, and activated by AMP, aspartate, 3-PGA, tryptophan and inorganic phosphate. ATP inhibited the enzyme competitively with respect to PEP and non-competitively with respect to ADP. Similarly, oxalate inhibition was also of competitive type with respect to PEP and non-competitive with respect to ADP. This inhibition by either ATP or oxalate was not due to chelation of Mg2+, as the inhibition was not relieved on increasing Mg2+ concentration even upto 30 mM. Initial velocity and product inhibition studies demonstrated the reaction mechanism to be compulsory ordered type. The enzyme seems to be regulated synergistically by ATP and citrate.