Inhibitory Concentrations Of ATP Research Articles

Kinetic Characterisation of Phosphofructokinase Purified fromSetaria cervi: A Bovine Filarial Parasite

Phosphofructokinase (PFK), a regulatory enzyme in glycolytic pathway, has been purified to electrophoretic homogeneity from adult female Setaria cervi and partially characterized. For this enzyme, the Lineweaver-Burk's double reciprocal plots of initial rates and D-fructose-6-phosphate (F-6-P) or Mg-ATP concentrations for varying values of cosubstrate concentration gave intersecting lines indicating that K m values for F-6-P (1.05 mM) and ATP (3 μM) were independent of each other. S. cervi PFK, when assayed at inhibitory concentration of ATP (>0.1 mM), exhibited sigmoidal behavior towards binding with F-6-P with a Hill coefficient (n) value equal to 1.8 and 1.7 at 1.0 and 0.33 mM ATP, respectively. D-fructose-1,6-diphosphate (FDP) competitively inhibited the filarial enzyme: K i and Hill coefficient values being 0.18 μM and 2.0, respectively. Phosphoenolpyruvate (PEP) also inhibited the enzyme competitively with the K i value equal to 0.8 mM. The Hill coefficient values (>1.5) for F-6-P (at inhibitory concentration of ATP) and FDP suggested its positive cooperative kinetics towards F-6-P and FDP, showing presence of more than one binding sites for these molecules in enzyme protein and allosteric nature of the filarial enzyme. The product inhibition studies gave us the only compatible mechanism of random addition process with a probable orientation of substrates and products on the enzyme surface.

Role of Ser530, Arg292, and His662 in the Allosteric Behavior of Rabbit Muscle Phosphofructokinase

Fructose-2,6-bisphosphate (Fru-2,6-P2) is a potent allosteric activator of the ATP-dependent phosphofructokinase (PFK) in eukaryotes. Based on the sequence homology between rabbit muscle PFK and two bacterial PFKs and the crystal structures of the latter, Ser530, Arg292 and His662 of the rabbit enzyme are implicated as binding sites for Fru-2,6-P2. We report here the effects of three mutations, S530D, R292A, and H662A on the activation of rabbit muscle PFK by Fru-2,6-P2. At pH 7.0 and the inhibitory concentrations of ATP, the native enzyme gives a classic sigmoidal response to changes in Fru-6-P concentration in the absence of Fru-2,6-P2 and a nearly hyperbolic response in the presence of the activator. Under the same conditions, no activation was seen for S530D. On the other hand, H662A can be activated but requires a 10-fold or higher concentration of Fru-2,6-P2. Limited activation was observed for mutant R292A. A model illustrating the sites for recognition of Fru-2,6-P2 in rabbit muscle PFK as well as the mechanism of allosteric activation is proposed.

Adenosine-Mediated Stimulation of Bone Cell Adenylate Cyclase Activity

Adenosine rapidly stimulated adenylate cyclase activity but did not modify cyclic AMP degradation when added to a particulate fraction prepared from isolated bone cells. The effect of adenosine was one-half maximal at 5-10 micronM, and was not mimicked by 5' AMP, inosine, guanosine, uridine, adenine, or ribose. Basal and adenosine-stimulated adenylate cyclase activites were directly proportional to the concentration of particulate protein in the assay system. Theophylline decreased the degree to which adenosine stimulated adenylate cyclase activity, whereas another phosphodiesterase inhibitor, RO-20-1724, failed to iiinfluence the effect of adenosine. Adenosine itself, and not a metabolite of adenosine is the stimulator of adenylate cyclase, since it was neither phosphorylated nor deaminated appreciably by the particulate fraction. The particulate fraction did not convert substrate ATP to adenosine in amounts sufficient to enhance adenylate cyclase. The stimulatory effect of adenosine was maximal at 1.2 mM Mg2+, declined with increases in the Mg2+ concentration, and was replaced by inhibition at 20 mM Mg2+. At 2.4 mM Mg2+, basal adenylate cyclase activity peaked at 1.1 mM ATP, and was inhibited by higher ATP concentrations. The magnitude of adenosine stimulation was greater at inhibitory concentrations of ATP than at concentrations which yielded maximum activity. The results suggest that the previously demonstrated ability of adenosine to increase cyclic 3'5' AMP levels in intact bone cells stems from its effect on adenylate cyclase. Adenosine may act by modifying the regulatory nfluence of free Mg2+, uncomplexed ATP, (or both), on adenylate cyclase. Theophylline appears to interfere with the action of adenosine by a mechanism which is distinct from its capacity to inhibit cyclic AMP phosphodiesterase activity. (Endocrinology 99:901,1976)

The effects of ammonium, inorganic phosphate and potassium ions on the activity of phosphofructokinases from muscle and nervous tissues of vertebrates and invertebrates.

1. The effect of NH4+, Pi and K+ on phosphofructokinase from muscle and nervous tissues of a large number of animals was investigated. The activation of the enzyme from lobster abdominal muscle by NH4+ was increased synergistically by the presence of Pi or SO4(2-). In the absence of K+, NH4+ plus Pi markedly activated phosphofructokinase from all tissues studied. In the presence of 100 mM-K+, NH4+ plus Pi activated phosphofructokinase from nervous tissue and muscle of invertebrates and the enzyme from brain of vertebrates, but there was no effect of NH4+ plus Pi on the enzyme from the muscles of vertebrates. Nonetheless, NH4+ plus Pi increased the activity of vertebrate muscle phosphofructokinase in the presence of 50 mM-K+ at inhibitory concentrations of ATP, i.e. these ions de-inhibited the enzyme. In the absence of NH4+ plus Pi, K+ activated phosphofructokinase from vertebrate tissues at non-inhibitory ATP concentrations, but the effect was less marked with the enzyme from invertebrate tissues. Indeed, high concentrations of K+ (greater than 50 mM) caused inhibition of invertebrate tissue phosphofructokinase. Of the other alkali-metal ions tested, only Rb+ activated phosphofructokinase from lobster abdominal muscle and rat heart muscle. 2. The properties of lobster abdominal-muscle phosphofructokinase were studied in detail. This muscle was chosen as representative of invertebrate muscle because large quantities of tissue could be obtained from one animal and the enzyme was considerably more stable in tissue extracts than in extracts of insect flight muscle. In general, the properties of the enzyme from this tissue were similar to those of the enzyme from many other tissues: ATP concentrations above an optimum value inhibited the enzyme and this inhibition was decreased by raising the fructose 6-phosphate or the AMP concentration. In particular, NH4+ plus Pi activated the enzyme at noninhibitory concentrations of ATP and they also relieved ATP inhibition (see above). 3. It is suggested that increases in the concentration of NH4+ and Pi, under conditions of increased ATP utilization in certain muscles and/or nervous tissue, may play a part in the stimulation of glycolysis through the effects on phosphofructokinase (the effect may be a direct activation and/or a relief of ATP inhibition). Changes in the concentration of NH4+ and Pi are consistent with this theory in nervous tissue and the anaerobic type of muscles. The role of AMP deaminase in production of NH4+ from AMP in these tissues is discussed in relation to the control of glycolysis.

Modulation of the Kinetic Properties of Phosphofructokinase by Ammonium Ions

Abstract Ammonium ions were shown to be much more efficient than potassium ions in activating rabbit skeletal muscle phosphofructokinase (EC 2.7.1.11). Under experimental conditions simulating physiological ones (pH 7.2, and inhibitory concentrations of ATP), the apparent dissociation constant of the phosphofructokinase-NH4+ complex was 0.33 mm. This molarity was shown to lie within the physiological concentration of ammonia in several tissues exhibiting pronounced glycolytic activity. The activation of the enzyme by NH4+ was very specific and rapid. It was observed also when ITP was the phosphate donor, indicating that the activation was not mere deinhibition of ATP. The form of phosphofructokinase predominant in the presence of NH4+ exhibited in a qualitative fashion the same allosteric characteristics of the enzyme form prevailing in its absence, i.e. sigmoidicity with respect to fructose 6-phosphate, and sensitivity to inhibition by citrate and higher concentrations of ATP. However, NH4+ (2 mm) decreased the Km for ATP (from 0.031 mm to 0.013 mm) and for fructose 6-phosphate (from 0.34 mm to 0.04 mm), and increased the Ki for citrate (from 0.025 mm to 0.055 mm) and for ATP (from 0.31 mm to 0.48 mm). It is proposed that the activation of phosphofructokinase by NH4+ could be a regulatory mechanism which provides the metabolites necessary for ammonia fixation and the maintenance of its concentration at tolerable levels. Moreover, the possibility that the increase in tissue concentrations of NH4+ under anoxia might be a contributing factor to the Pasteur effect is discussed.

Purification and regulatory properties of ATP-sensitive phosphofructokinase from yeast.

Phosphofructokinase from baker's yeast has been purified 370‐fold. The purified enzyme is homogeneous as judged by analytical ultracentrifugation and disc electrophoresis. It exhibits the kinetic properties reported for phosphofructokinase from yeast and other sources. The purified enzyme is very sensitive to inhibition by ATP. The inhibition is pH‐dependent with a maximum around pH 7.5.The influence of different effectors on the enzymatic activity and on the inhibition by ATP are described. NH4+ ions cause a general stimulation of enzymatic activity, while the sensitivity to ATP is abolished. ADP inhibits the enzyme at non‐inhibitory ATP levels and stimulates the activity at inhibitory concentrations of ATP. Phosphoenolpyruvate is a negative effector. Inorganic phosphate inhibits the activity by competition with Fru‐6‐P and increases the inhibitory effect of ATP.