Allosteric enzymes phosphofructokinase 1 activation

Phosphofructokinase possesses two substrates, ATP and F6P, which it transforms into ADP and FBP. A complete model for this reaction should therefore take into account the evolution of these four metabolites. However, studies carried out in yeast indicate that the couple ATP-ADP plays a more important role than the couple F6P-FBP in the control of oscillations. Indeed, the addition of ADP ehcits an immediate phase shift of the oscillations (fig. 2.8) while the effect of FBP is much weaker (Hess Boiteux, 1968b Pye, 1969). The predominant regulation is thus exerted by ADP. In order to keep the model as simple as possible and to limit the number of variables to only two, which allows us to resort to the powerful tools of phase plane analysis, the situation in which an allosteric enzyme is activated by its unique reaction product is considered (fig. 2.10). This monosubstrate, product-activated. 

Figure 6.24 The function of the enzyme phosphofructokinase. (a) Phosphofructokinase is a key enzyme in the gycolytic pathway, the breakdown of glucose to pyruvate. One of the end products in this pathway, phosphoenolpyruvate, is an allosteric feedback inhibitor to this enzyme and ADP is an activator, (b) Phosphofructokinase catalyzes the phosphorylation by ATP of fructose-6-phosphate to give fructose-1,6-bisphosphate. (c) Phosphoglycolate, which has a structure similar to phosphoenolpyruvate, is also an inhibitor of the enzyme.

A typical chemical system is the oxidative decarboxylation of malonic acid catalyzed by cerium ions and bromine, the so-called Zhabotinsky reaction this reaction in a given domain leads to the evolution of sustained oscillations and chemical waves. Furthermore, these states have been observed in a number of enzyme systems. The simplest case is the reaction catalyzed by the enzyme peroxidase. The reaction kinetics display either steady states, bistability, or oscillations. A more complex system is the ubiquitous process of glycolysis catalyzed by a sequence of coordinated enzyme reactions. In a given domain the process readily exhibits continuous oscillations of chemical concentrations and fluxes, which can be recorded by spectroscopic and electrometric techniques. The source of the periodicity is the enzyme phosphofructokinase, which catalyzes the phosphorylation of fructose-6-phosphate by ATP, resulting in the formation of fructose-1,6 biphosphate and ADP. The overall activity of the octameric enzyme is described by an allosteric model with fructose-6-phosphate, ATP, and AMP as controlling ligands. 

Allosteric Enzymes Typically Exhibit a Sigmoidal Dependence on Substrate Concentration The Symmetry Model Provides a Useful Framework for Relating Conformational Transitions to Allosteric Activation or Inhibition Phosphofructokinase Allosteric Control of Glycolysis Is Consistent with the Symmetry Model Aspartate Carbamoyl Transferase Allosteric Control of Pyrimidine Biosynthesis Glycogen Phosphorylase Combined Control by Allosteric Effectors and Phosphorylation... 

The same protein kinase that phosphorylates glycogen phosphorylase and glycogen synthase does not phosphorylate the enzymes of pseudocycle II. Rather an enzyme gets phos-phorylated that catalyzes the synthesis of a potent allosteric effector of the two relevant enzymes, phosphofructokinase and fructose bisphosphate phosphatase. In the liver the un-phosphorylated form this enzyme synthesizes fructose-2,6-bisphosphate. Phosphorylation converts it into a degradative enzyme for the same compound. Fructose-2,6-bisphosphate is an activator of phosphofructokinase and an inhibitor of fructose bisphosphate phosphatase. As a result the net effect of glucagon on pseudocycle II is to stimulate fructose bisphosphate phosphatase while inhibiting phosphofructokinase (see table 12.2 and fig. 12.30). 

It is phosphofructokinase, which is an allosteric enzyme thus, its activity is regulated by a number of effectors (Chap. 10), all of which are involved in energy transduction. 

The enzyme phosphofructokinase is allosteric, that is, it is made up of equivalent units that possess specific reaction sites for the fixation of the substrate and product. Each unit exists in two conformational states one active with more affinity for the substrate, and one inactive. The reaction products of phosphofructokinase (FDP and ADP) displace the conformational equilibrium in favor of the active form of the enzyme. This may create a destabilizing effect on the excess entropy production. In the glycolytic cycle, the allosteric properties of the phosphofructokinase may lead to oscillations. Consider the following simple model... 

C. The activity of regulatory enzymes such as fructose-1,6-bisphos-phatase, hexokinase, phosphofructokinase 1, and pyruvate kinase are frequently controlled by binding allosteric effectors. These allosteric enzymes usually exhibit sigmoidal kinetics. Lactate dehydrogenase is not controlled by allosteric effectors and therefore would be expected to exhibit Michaelis-Menten kinetics. 

Phosphofructokinase activity is sensitive to both positive and negative allosterism. For instance, when ATP is present in abundance, a signal that the body has sufficient energy, it binds to an effector binding site on phosphofructokinase. This inhibits the activity of the enzyme and, thus, slows the entire pathway. An abundance of AMP (adenosine monophosphate), which is a precursor of ATP, is evidence that the body needs to make ATP to have a sufficient energy supply. When AMP binds to an effector binding site on phosphofructokinase, enzyme activity is increased, speeding up the reaction and the entire pathway. 

Phosphofructokinase, the enzyme that catalyzes the third reaction in glycolysis, is a key regulatory enzyme in the pathway. ATP is an allosteric inhibitor of phosphofructokinase, whereas AMP and ADP are allosteric activators. Another allosteric inhibitor of phosphofructokinase is citrate. As we will see in the next chapter, citrate is the first intermediate in the citric acid cycle. The citric acid cycle is a pathway that results in the complete oxidation of the pyruvate produced by glycolysis. A high concentration of citrate signals that sufficient substrate is entering the citric acid cycle. The inhibition of phosphofructokinase by citrate is an example of feedback inhibition the product, citrate, allosterically inhibits the activity of an enzyme early in the pathway. 

This reaction is catalyzed by the enzyme fructose-l,6-hisphosphatase, an allosteric enzyme strongly inhibited by adenosine monophosphate (AMP) but stimulated by ATP. Because of allosteric regulation, this reaction is also a control point in the pathway. When the cell has an ample supply of ATP, the formation rather than the breakdown of glucose is favored. This enzyme is inhibited by fructose-2,6- >t5phosphate, a compound we met in Section 17.2 as an extremely potent activator of phosphofructokinase. We shall return to this point in the next section. 

High intracellular concentrations of fructose 6-phosphate activate a second enzyme, phosphofructokinase-2, which catalyses the synthesis of fructose 2,6-bisphosphate from fructose 6-phosphate (Figure 10.4). Fructose 2,6-bisphosphate is an allosteric activator of phosphofructokinase and an allosteric inhibitor of fructose 1,6-bisphosphatase. It thus acts to both increase glycolysis and inhibit gluconeogenesis. This is feed-forward control — allosteric activation of phosphofructokinase because there is an increased concentration of substrate available. 

Within glycolysis, the main allosteric control is exercised by phosphofructokinase, a complicated enzyme unusual in that its activity is stimulated by one of its products (ADP) and inhibited by one of its substrates (ATP). One further point about this enzyme which will be important to us later, in Aspergillus spp., elevated levels of ammonium ions relieve phosphofructokinase of inhibition by titrate. 

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