Phosphofructokinase, inhibition

Glycolysis and the citric acid cycle (to be discussed in Chapter 20) are coupled via phosphofructokinase, because citrate, an intermediate in the citric acid cycle, is an allosteric inhibitor of phosphofructokinase. When the citric acid cycle reaches saturation, glycolysis (which feeds the citric acid cycle under aerobic conditions) slows down. The citric acid cycle directs electrons into the electron transport chain (for the purpose of ATP synthesis in oxidative phosphorylation) and also provides precursor molecules for biosynthetic pathways. Inhibition of glycolysis by citrate ensures that glucose will not be committed to these activities if the citric acid cycle is already saturated. 

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. 

A decreased glycolytic rate has been proposed as a cause of muscle fatigue and related to pH inhibition of glycolytic enzymes. Decreasing pH inhibits both phosphorylase kinase and phosphofructokinase (PFK) activities. PFK is rate determining for glycolytic flux and therefore must be precisely matched to the rate of ATP expenditure. The essential characteristic of PFK control is allosteric inhibition by ATP. This inhibition is increased by H and PCr (Storey and Hochachka, 1974 ... 

The most potent positive allosteric effector of phospho-ffuctokinase-1 and inhibitor of fructose-1,6-bisphos-phatase in liver is fructose 2,6-bisphosphate. It relieves inhibition of phosphofructokinase-1 by ATP and increases affinity for fructose 6-phosphate. It inhibits fructose-1,6-bisphosphatase by increasing the for fructose 1,6-bisphosphate. Its concentration is under both substrate (allosteric) and hormonal control (covalent modification) (Figure 19-3). 

Fructose 2,6-bisphosphate is formed by phosphorylation of fructose 6-phosphate by phosphofructoki-nase-2. The same enzyme protein is also responsible for its breakdown, since it has fructose-2,6-hisphos-phatase activity. This hifrmctional enzyme is under the allosteric control of fructose 6-phosphate, which stimulates the kinase and inhibits the phosphatase. Hence, when glucose is abundant, the concentration of fructose 2,6-bisphosphate increases, stimulating glycolysis by activating phosphofructokinase-1 and inhibiting... 

Secondary signals Glucose signals activate (fructose 2,6-bis-phosphate activates phosphofructokinase). Low-glucose signals inhibit. 

Figure 30. A medium complexity model of yeast glycolysis [342], The model consists of nine metabolites and nine reactions. The main regulatory step is the phosphofructokinase (PFK), combined with the hexokinase (HK) reaction into a single reaction vi. As in the minimal model, we only consider the inhibition by its substrate ATP, although PFK is known to have several effectors. External glucose (Glc ) and ethanol (EtOH) are assumed to be constant. Additional abbreviations Glucose (Glc), fructose 1,6 biphosphate (FBP), pool of triosephosphates (TP), 1,3 biphosphogly cerate (BPG), and the pool of pyruvate and acetaldehyde (Pyr).

Phosphofructokinase, the activity of which is inhibited by ATP and phosphocreatine, but this inhibition is relieved by AMP and phosphate. 

Figure 16.1 The glucose/fatty add cycle. The dotted Lines represent regulation. Glucose in adipose tissue produces glycerol 3-phosphate which enhances esterification of fatty acids, so that less are available for release. The effect is, therefore, tantamount to inhibition of lipolysis. Fatty acid oxidation inhibits pyruvate dehydrogenase, phosphofructokinase and glucose transport in muscle (Chapters 6 and 7) (Randle et al. 1963).

Fructose 2,6-bisphosphate (Fru-2,6-bP) plays an important part in carbohydrate metabolism. This metabolite is formed in small quantities from fructose 6-phosphate and has purely regulatory functions. It stimulates glycolysis by allosteric activation of phosphofructokinase and inhibits gluconeogenesis by inhibition of fructose 1,6-bisphosphatase. 

Fig. 2.6. Regulation of Phosphofructokinase from Bacillus stearothermophilus. The tetrameric phosphofructokinase is aUostericaUy regulated by ADP, Frc-6-phosphate, and phosphoenolpyruvate (PEP). The binding of ADP and Frc-6-phosphate converts the enzyme into the active R state. PEP binds to the T state and inhibits phosphofructokinase. The circles represent the R state, and the squares represent the T state of the enzyme.

Phosphofructokinase-1 is a regulatory enzyme (Chapter 6), one of the most complex known. It is the major point of regulation in glycolysis. The activity of PFK-1 is increased whenever the cell s ATP supply is depleted or when the ATP breakdown products, ADP and AMP (particularly the latter), are in excess. The enzyme is inhibited whenever the cell has ample ATP and is well supplied by other fuels such as fatty acids. In some organisms, fructose 2,6-bisphosphate (not to be confused with the PFK-1 reaction product, fructose 1,6-bisphosphate) is a potent allosteric activator of PFK-1. The regulation of this step in glycolysis is discussed in greater detail in Chapter 15. 

Phosphofructokinase-1 is also inhibited by citrate, the first intermediate of the citric acid cycle. When the cycle is idling, citrate accumulates within mitochondria, then spills into the cytosol. When the concentrations of both ATP and citrate rise, they produce a concerted allosteric inhibition of phosphofructokinase-1 that is greater than the sum of their individual effects, slowing glycolysis. 

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