Formation Of Phosphoenolpyruvate Research Articles

Stimulation of phosphoenolpyruvate formation by oleic acid and other uncouplers of oxidative phosphorylation in rabbit liver mitochondria

Oleic acid, like DNP, stimulates the oxidation of α-ketoglutarate and pyruvate by rabbit liver mitochondria, and at the same time accelerates the formation of phosphoenolpyruvate from these substrates. The net production of malate, citrate and acetoacetate similarly depends on the concentration of uncoupler.

The direct synthesis of phosphoenolpyruvate from pyruvate by Escherichia coli.

Extracts ofEscherichia coliare shown to contain an enzyme system which in the presence of Mg2+catalyses the direct formation of phosphoenolpyruvate from pyruvate andATPwith concomitant formation ofAMPand inorganic phosphate. This enzyme, which has been designated 'phosphoenolpyruvate synthase' (PEP-synthase) has been purified 80-fold and is free of pyruvate kinase activity;PEPsynthesis proceeded most rapidly at pH 8 to 8.5. At pH values between 6.2 and 7.5 the enzyme can catalyse the formation ofATPand pyruvate fromPEP,AMPand inorganic phosphate; if arsenate is used instead of phosphate, pyruvate andADPare produced instead. Studies of the enzymic formation ofPEPwithATPspecifically labelled with32P, and of the reverse reaction with [U-14C]AMP, suggest that thePEP-synthase reaction involves the transfer of a pyrophosphoryl-group. The physiological role ofPEP-synthase has been demonstrated with mutants of E.colidevoid of the enzyme: in contrast to wild-type organisms, such mutants neither grow on pyruvate, lactate or alanine, nor form glycogen from lactate. It is thus concluded thatPEP-synthase plays an important role in the anaplerotic and the biosynthetic reactions which enable the organisms to grow on pyruvate as sole carbon source.

The regulation of phosphoenolpyruvate synthesis in pigeon liver.

1. The intracellular location and maximal activities of enzymes involved in phosphoenolpyruvate synthesis have been investigated in pigeon liver. Enolase and pyruvate kinase were cytoplasmic, and the activities were 50-60 and 180-210mumoles/min./g. dry wt. at 25 degrees respectively. Phosphoenolpyruvate carboxykinase was present exclusively, and nucleoside diphosphokinase predominantly, in the mitochondria; the particles had to be disrupted to elicit maximal activities, which were 27-33 and 400-600mumoles/min./g. dry wt. at 25 degrees respectively. The activities of all four enzymes did not change significantly during 48hr. of starvation. 2. Conditions for incubation of washed isolated mitochondria were established, to give high rates of synthesis of phosphoenolpyruvate, linear with time and proportional to mitochondrial concentration. Inorganic phosphate and added adenine nucleotides were stimulatory, whereas added Mg(2+) inhibited, partly owing to activation of contaminant pyruvate kinase. Phosphoenolpyruvate formation occurred from oxaloacetate, malate, fumarate, succinate, alpha-oxoglutarate and citrate, in decreasing order of effectiveness. 3. The steady-state ATP/ADP ratio of mitochondrial suspensions was decreased in the presence of added 2.5mm-Mg(2+) (owing to stimulation of adenylate kinase and possibly of an adenosine triphosphatase), 0.5mm-Ca(2+) or 0.4mm-dinitrophenol. In each case the rate of substrate removal and oxygen uptake was increased, whereas phosphoenolpyruvate synthesis was inhibited. Citrate formation was enhanced, owing to de-inhibition of citrate synthase. These effects were not primarily related to changes in the oxaloacetate concentration. 4. Both phosphoenolpyruvate carboxykinase and nucleoside diphosphokinase were active within the atractylosidesensitive barrier to the mitochondrial metabolism of added adenine nucleotides. There was no correlation between the rate of substrate-level phosphorylation associated with the oxidation of alpha-oxoglutarate, and the synthesis of phosphoenolpyruvate. 5. The results suggest that phosphoenolpyruvate formation in pigeon-liver mitochondria is regulated partly by the phosphorylation state of the adenine and guanine nucleotides, and partly by variations in the oxaloacetate concentration, all in the mitochondrial matrix. 6. Phosphoenolpyruvate is assumed to be the metabolite transported from the mitochondria to the cytoplasm during gluconeogenesis from oxaloacetate in pigeon liver.

Control of Glycolysis in the Human Red Blood Cell

Abstract Red cells are known to use glucose and produce lactate at an increased rate in the presence of increased inorganic phosphate. The stimulation by inorganic phosphate of hexokinase and phosphofructokinase is recognized, but the cause of the necessary rate increase of pyruvate kinase is not. Kinetic studies with this enzyme from normal and human mutant cells and steady state studies with whole glycolyzing cells support the conclusion that pyruvate kinase is normally operating far below saturation with respect to phosphoenolpyruvate. The increase in the rate of formation of phosphoenolpyruvate is primarily responsible for the increase in its rate of reaction in the kinase step. Pyruvate kinase-deficient cells with about 10% as much enzyme activity reach much higher and even saturating levels of phosphoenolpyruvate in order to utilize a larger proportion of their enzyme. This is necessary to achieve the normal glycolytic rates observed. The explanation for the accumulation of fructose diphosphate and dihydroxyacetone phosphate at high glycolytic rates in normal and deficient cells is that glyceraldehyde phosphate dehydrogenase becomes ratelimiting as a result of an increased ratio of reduced to oxidized nicotinamide adenine dinucleotide and increased 1,3-diphosphoglyceric acid. These factors increase at high glycolytic rate because the increased phosphoenolpyruvate plus phosphoglyceric acids leave an amount of reduced nicotinamide adenine dinucleotide unoxidized and because the phosphoglycerate kinase reaction is probably at equilibrium. The large pool of 2,3-diphosphoglyceric acid in many mammalian red cells is seen as a possible source of the reduced nicotinamide adenine dinucleotide oxidant, pyruvate, necessary to regulate this effect which, in the limit, would exhaust the cell's adenosine triphosphate supply.

Net formation of phosphoenolpyruvate from pyruvate by Escherichia coli

Net formation of phosphoenolpyruvate from pyruvate by Escherichia coli

Biosynthesis of glucose from pyruvate-2-C 14 and aspartate-3-C 14 in a mollusc

1. 1. The clam, Rangia cuneata Gray, is capable of synthesizing glucose from aspartate-3-C 14 and pyruvate-2-C 14 in foot muscle, hepatopancreas and mantle. 2. 2. In muscle and hepatopancreas the isotope distribution in the glucose (from glycogen) with aspartate-3-C 14 as the precursor is compatible with a mechanism in which the isotope is completely randomized before formation of phosphoenolpyruvate. In mantle this randomization is incomplete. 3. 3. The isotope distribution in the glucose (from glycogen) with pyruvate-2-C 14 as a precursor is compatible with the operation of two pathways, viz. the direct conversion of pyruvate to phosphoenolpyruvate and the carboxylation of pyruvate to oxalacetate prior to phosphoenolpyruvate formation. The latter mechanism was found to be the predominant pathway in the three organs studied. 4. 4. The predominance of the “indirect pathway” for glucose synthesis from pyruvate is correlated with high levels of PEP-carboxykinase activity in these three organs.

On the nature of the compartmentation of the substrate-linked phosphorylation in rat-liver mitochondria

Summary 1 During the oxidation of α-oxoglutarate or glutamate by rat-liver mitochondria in the presence of dinitrophenol and absence of added inorganic phosphate and phosphate acceptor the endogenous inorganic phosphate is quantitatively converted to phosphoenolpyruvate. The depletion of inorganic phosphate results in a decline in the rate of respiration. 2 The conversion of inorganic phosphate to phosphoenolpyruvate during α-oxoglutarate oxidation is inhibited by malonate. Under these conditions, α-oxoglutarate disappearance is stimulated. If malate is added as well as malonate, phosphoenolpyruvate formation is restored, and α-oxoglutarate disappearance is diminished. 3 In rat-heart sarcosomes, which contain no phosphopyruvate carboxylase (EC 4.1.1.32), the rate of oxidation of α-oxoglutarate in the presence of dinitrophenol and absence of phosphate acceptor does not show the marked decline observed with liver mitochondria. Inorganic phosphate does not disappear during the incubation.

The endogenous formation of phosphoenolpyruvate by rat-liver mitochondria

The endogenous formation of phosphoenolpyruvate by rat-liver mitochondria

THE EFFECT OF 2,4-DINITROPHENOL ON THE FORMATION OF PHOSPHOENOLPYRUVATE BY LIVER MITOCHONDRIA

THE EFFECT OF 2,4-DINITROPHENOL ON THE FORMATION OF PHOSPHOENOLPYRUVATE BY LIVER MITOCHONDRIA