Apparent Km For Phosphoenolpyruvate Research Articles

Purification and characterization of pyruvate kinase from lamprey ( Entosphenus japonicus) muscle

Pyruvate kinase from skeletal muscle of lamprey ( Entosphenus japonicus), which is one of the most primitive living vertebrates, has been purified by approxImately 110-fold. The isolation procedure includes chromatography on Phosphocellulose, Phenyl-5PW, and Sephacryl S-300. Sodium dodecyl sulfate gel electrophoresis shows 59 000 as the deduced subunit molecular weight and gel filtration shows 232 000 as the tetramer of the subunits. The apparent Km for phosphoenolpyruvate and ADP are 0.41 mM and 0.31 mM at pH 7.4, respectively, when the purified enzyme is saturated with the second substrate. When the enzyme is activated in the presence of fructose-1,6-diphosphate, the Km for PEP changes to 0.087 mM, and the Hill coefficient changes from 1.3 to 0.98.

The role of phosphoenolpyruvate in the simultaneous uptake of fructose and 2-deoxyglucose by Escherichia coli.

Nonmetabolizable glucose analogs inhibit the growth of Escherichia coli on a wide variety of carbon sources. This phenomenon was investigated with particular reference to the effect of 2-deoxyglucose (2DG) on growth on fructose as sole carbon source. When the inhibitor is supplied in sufficiently low concentrations, the initial arrest of growth is overcome; this relief of inhibition is aided by means that increase the availability of phosphoenolpyruvate (PEP) to the growing cells, such as the use of L-aspartate instead of ammonium chloride as sole nitrogen source for growth, and the introduction of the pps+ allele into a pps- strain. Studies with [14C]2DG showed that the analog or its 6-phosphate as such did not inhibit growth but that 2DG exerted its effect by competing for intracellular PEP and lowering its concentration below that needed to sustain growth. Direct measurements of the PEP-dependent phosphorylation of 2DG and of fructose by permeabilized E. coli showed that the apparent Km for PEP was nearly 7 times higher for 2DG that it was for fructose, although the apparent Vmax for 2DG was nearly 3 times that for fructose; this explains the ability of cells to overcome the inhibition by low, but not by high, concentrations of 2DG.

Identification of the reactive cysteines of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase and their nonessentiality for enzymatic catalysis.

Reaction of 5-enolpyruvylshikimate-3-phosphate synthase of Escherichia coli with the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) leads to a modification of only 2 of the 6 cysteines of the enzyme, with a significant loss of its enzymatic activity. Under denaturing conditions, however, all 6 cysteines of 5-enolpyruvylshikimate-3-phosphate synthase react with DTNB, indicating the absence of disulfide bridges in the native protein. In the presence of shikimate 3-phosphate and glyphosate, only 1 of the 2 cysteines reacts with the reagent, with no loss of activity, suggesting that only 1 of these cysteines is at or near the active site of the enzyme. Cyanolysis of the DTNB-inactivated enzyme with KCN leads to elimination of 5-thio-2-nitrobenzoate, with formation of the thiocyano-enzyme. The thiocyano-enzyme is fully active; it exhibits a small increase in its I50 for glyphosate (6-fold) and apparent Km for phosphoenolpyruvate (4-fold) compared to the unmodified enzyme. Its apparent Km for shikimate 3-phosphate is, however, unaltered. These results clearly establish the nonessentiality of the active site-reactive cysteine of E. coli 5-enolpyruvylshikimate-3-phosphate synthase for either catalysis or substrate binding. Perturbations in the kinetic constants for phosphoenolpyruvate and glyphosate suggest that the cysteine thiol is proximal to the binding site for these ligands. By N-[14C]ethylmaleimide labeling, tryptic mapping, and N-terminal sequencing, the 2 reactive cysteines have been identified as Cys408 and Cys288. The cysteine residue protected by glyphosate and shikimate 3-phosphate from its reaction with DTNB was found to be Cys408.

Multiple interaction of fructose 1,6-bisphosphate and other effectors on phosphoenolpyruvate carboxylase from Brevibacterium flavum and its aspartate-producing mutant.

Phosphoenolpyruvate (PEP) carboxylase purified from Brevibacterium flavum was specifically activated by fructose 1, 6-bisphosphate (FBP). The other intermediates of sugar metabolism or their structural analogues did not influence the activity. FBP decreased the apparent Km for PEP but did not affect that for another substrate, bicarbonate, or the apparent maximum velocity for PEP. The dissociation constants for FBP from enzyme-FBP and enzyme-PEP-FBP complex were 63 and 32 μM, respectively, being almost equivalent to those for acetyl-CoA. Synergistic activation by FBP and acetyl-CoA was not observed with the B. flavum enzyme, unlike the Escherichia coli enzyme. FBP, like acetyl-CoA, was kinetically competitive with aspartate. With respect to another feedback inhibitor, 2-oxoglutarate, acetyl-CoA was non-competitive, whereas FBP was of mixed-type, i.e., FBP but not acetyl-CoA prevented 2-oxoglutarate from binding to the enzyme to a certain extent. Homotropic cooperativity was observed only with FBP but not with acetyl-CoA in the absence of inhibitors. Cooperativities of FBP and acetyl-CoA were increased by aspartate but not by 2-oxoglutarate. In the aspartate-overproducing mutant enzyme, the Michaelis constant for PEP was decreased, whereas the inhibitor constant for aspartate with or without simultaneous addition of 2-oxoglutarate and the activator constants for FBP and acetyl-CoA were increased. The decreased Michaelis constant for PEP was comparable to the apparent Km of the wild-type enzyme for PEP in the presence of the saturated concentration of FBP, and would result in a further decrease in the affinity of the mutant enzyme for aspartate.

Thermodynamic linkages in rabbit muscle pyruvate kinase: kinetic, equilibrium, and structural studies.

The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23 degrees C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23 degrees C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].

Hepatic L-type pyruvate kinase: separation of unphosphorylated, phosphorylated and proteolytically modified in vivo forms.

After partial chromatographic purification of rat liver cell sap on DEAE-cellulose, including the removal of type M2 pyruvate kinase, different forms of type L pyruvate kinase were separated by chromatofocusing. Three fractions of pyruvate kinase activity were found, eluting at pH 5.0, 5.2, and 5.3, respectively. The first one was identified as phosphorylated and the second one as unphosphorylated pyruvate kinase. There were strong indications that the third fraction represented a proteolytically modified form of the enzyme, since it co-migrated with a form modified in vitro and had a similarly increased apparent Km for phosphoenolpyruvate. To rule out the possibility of this being a phosphorylated form of pyruvate kinase, the enzyme was incubated with a phosphoprotein phosphatase and then phosphorylated with cAMP-dependent protein kinase. The enzyme was not phosphorylated, like pyruvate kinase modified with subtilisin or calcium-activated protease. There is some evidence that a proteolytically modified pyruvate kinase exists in vivo. This enzyme form has not previously been demonstrated in cell sap, prior to exposure to proteolytic enzymes. The relative amounts of the three forms were determined in livers from starved rats and rats fed on a normal or a carbohydrate-rich diet.

Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli.

3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase has been purified 450-fold from frozen Escherichia coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+, and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to be 45 degrees C, and the energy of activation calculated by the Arrhenius equation was 15,000 calories (ca. 3,585 J) per mol. The enzyme activity was shown to be pH and buffer dependent, showing two pH optima, one at pH 4.0 to 6.0 in succinate buffer and one at pH 9.0 in glycine buffer. The isoelectric point of the enzyme was 5.1. KDO-8-phosphate synthetase had a molecular weight of 90,000 +/- 6,000 as determined by molecular sieving through G-200 Sephadex and by Ferguson analysis using polyacrylamide gels. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 90,000-molecular-weight native enzyme was composed of three identical subunits, each with an apparent molecular weight of 32,000 +/- 4,000. The enzyme had an apparent Km for D-arabinose-5-phosphate of 2 X 10(-5) M and an apparent Km for PEP of 6 X 10(-6) M. No other sugar or sugar-phosphate could substitute for D-arabinose-5-phosphate. D-Ribose-5-phosphate was a competitive inhibitor of D-arabinose-5-phosphate, with an apparent Ki of 1 X 10(-3) M. The purified enzyme has been utilized to synthesize millimole quantities of pure KDO-8-phosphate.

Purification and Characterization of Phosphoenolpyruvate Carboxylase from Maize Leaves

Phosphoenolpyruvate carboxylase has been purified to homogeneity from maize (Zea mays L. var. Golden Cross Bantam T51) leaves. The ratio of specific activities in crude extracts and the purified enzyme suggests that the enzyme is a major soluble protein in the tissue. The enzyme has a sedimentation coefficient (s(20,w)) of 12.3S and a molecular weight, determined by sedimentation equilibrium, of 400,000 daltons. Dissociation of the enzyme and electrophoresis on dodecyl sulfate polyacrylamide gels yields a single stained band which corresponds to a subunit weight of 99,000 daltons. Thus it appears that the native enzyme is composed of four identical or similar polypeptide chains.The enzyme yields cooperative rate-concentration plots (Hill number of 2) with phosphoenolpyruvate as the variable substrate at pH 7. This cooperativity disappears in the presence of an activator, glucose-6-P, or by raising the pH of the assay mixture to 8. Glycerol (20%, v/v) exerts a similar effect. The enzyme is also activated in the presence of glycine which causes an increase in V(max) without significant effect on the apparent Km for phosphoenolpyruvate and Hill number. The apparent Km for HCO(3) (-) is 0.02 mm, and the activation constant for Mg(2+) is 1.54 mm at pH 7. There is an abrupt discontinuity in Arrhenius plots and an associated increase in activation energy below 10.8 C.

Inhibition of phosphoenolpyruvate carboxylase from C4 plants by malate and aspartate

The activity of phosphoenolpyruvate carboxylase in leaf extracts of C4 plants has been shown to be inhibited by the C4 acids malate and aspartate. The magnitude of inhibition observed with phosphoenolpyruvate carboxylase from Digitaria sanguinalis leaves was dependent on the pH of the assay mixture and the concentration of phosphoenolpyruvate (PEP), Mg2+. and the C4 acid inhibitor. The percentage inhibition decreased with increasing pH up to 8.5. At rate-limiting concentrations of phosphoenolpyruvate the pH optimum of the enzyme was 7.8, whereas the enzyme partially inhibited by malate or aspartate had a pH optimum of roughly 8.3. Malate and aspartate inhibited the enzyme by increasing the apparent Km for phosphoenolpyruvate without altering the maximum velocity, such that inhibition was greatest at rate-limiting concentrations of PEP but was not observed at saturating (3 mM) substrate levels. Glucose-6-P. an activator of PEP carboxylase, decreased the apparent Km for PEP and reversed the inhibition by malate and aspartate. Inhibition was also dependent on the concentration of Mg2+. At 1 mM Mg2+, 1 mM malate caused 60% inhibition, but at 5 mM Mg2+ the inhibition was reduced to 15%. Mg2+ at concentrations from 1 to 5 mM had no effect on the apparent Km for PEP. With Sephadex extracts of PEP carboxylase from leaves of D. sanguinalis, malate and aspartate were equally inhibitory, with roughly 2 mM levels required for 50% inhibition. Both malate and aspartate were inhibitory with PEP carboxylase extracted from leaves of a variety of C4∙ but not C3∙ plants. The significance of the observed inhibition and activation of PEP carboxylase by metabolites is discussed in relation to C4 photosynthesis.

Kinetic studies on the regulation of rabbit liver pyruvate kinase

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(-)/ADP(2-) 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 Mg(2+) 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 (n(H)=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 K(m) 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 (K(m)=0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg(2+).

Some Kinetic Properties of Liver Pyruvate Kinase (Type L)

Abstract Type L pyruvate kinase from mouse liver has been studied. The enzyme is strongly inhibited by low concentrations of copper ions, and the inhibition is reversed by fructose 1,6-diphosphate. In the absence of inhibitor, fructose 1,6-diphosphate also increases the pyruvate kinase activity at pH values higher than 7. Furthermore, the Cu++ inhibition is pH and K+ concentration dependent. Plots of reaction rates against phosphoenolpyruvate concentration give a sigmoid curve whereas a hyperbolic curve is obtained for the other substrate (adenosine diphosphate). When fructose 1,6-diphosphate is added, the response to phosphoenolpyruvate concentration is transformed to give a Michaelian curve and the affinity of the enzyme for this substrate increases. In the presence of Cu++, the Km for adenosine diphosphate is not substantially modified, but the Vmax decreases markedly. On the other hand, the apparent Km for phosphoenolpyruvate is considerably increased by this cation. The enzyme has a full requirement for Mg++ and a monovalent cation (K+ or NH4+). The activation of the enzyme with NH4+ has been found to be more efficient than with K+. The physiological role of the Cu++-fructose 1,6-diphosphate interrelation in the balance between glycolysis and gluconeogenesis is discussed.