Phosphofructokinase, enzymic activity

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. 

FIGURE 19.9 Fructose-2,6-bisphosphate activates phosphofructokinase, iucreasiug the affinity of the enzyme for fructose-6-phosphate and restoring the hyperbolic dependence of enzyme activity on substrate. 

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 ... 

To give rise to oscillatory behavior instead of a biochemical explosion, selfamplification must, however, be coupled to a limiting process. Such a limiting process can be viewed as a form of negative feedback because it occurs as a consequence of the positive feedback that precedes it. Thus, in the case of glycolytic oscillations, the activation of phosphofructokinase by a reaction product is followed by a counteracting fall in the rate of the enzymatic reaction, due to the enhanced substrate consumption associated with enzyme activation. In Ca + pulsatile signaling, the explosive rise in cytosolic Ca + due... 

The third system is that of thioltransferase, which may not be distinct from glutaredoxin in some tissues. The thioltransferase system is composed of thioltransferase, GSH, glutathione reductase and NADPH. All thioltransferases which have been found in mammalian cells have molecular weights which are close to 11 KDa. Sub-millimolar concentrations of either cystamine or cystine inactivate GST-IT from human placenta with concomitant formation of enzyme mixed-disulfides [282]. Thioltransferase from human placenta specifically reactivates GST-IT in the presence of 10-100 pM GSH while thioredoxin is inactive [282], Thioltransferase-mediated cleavage of mixed disulfides was also shown to restore enzyme activities in phosphofructokinase [283] and pyruvate kinase [284]. 

Phosphofructokinase deficiency (OMIM 171850) is a rare autosomal recessively inherited disorder. Since red cells contain both M and L submits, mutations affecting the genes coding for these subunits wfil affect enzyme activity. Thus when the PFK deficiency mutation affects only the L subunit, RBCs have only M4 and are PFK deficient. In such cases, there is a mild hemolytic disorder without myopathy. However, when the M subunit is deficient, muscle PFK is virtually absent together with partial PFK deficiency in RBCs, which wfil have only L. Therefore deficiency of the M subunit causes myopathy and a mild hemolytic disorder. 

The mechanism for regulation of phosphofructokinase in other tissues has proved to be one of controlling the rate of enzyme activity by varying the binding to its substrate, D-fructose 6-phosphate. The solubilized enzyme from rhesus-monkey spermatozoa is found to pos-... 

The last enzyme in glycolysis, pyruvate kinase, is also subject to allosteric regulation. In this case, fructose-l,6-bisphosphate is the allosteric activator. It is interesting that fructose-l,6-bisphosphate is the product of the reaction catalyzed by phosphofructokinase. Thus, activation of phosphofructokinase results in the activation of pyruvate kinase. This is an example of feedforward activation because the product of an earlier reaction causes activation of an enzyme later in the pathway... 

The function of magnesium in enzyme activity may either be to form a complex with the substrate, as in the magnesium-ATP complex formed in creatine kinase and phosphofructokinase, or to bind to the enzyme and either produce an allosteric activation or play a direct role in catalysis. If an enzyme is known to utilize a nucleotide as one of its substrates, it can be assumed that magnesium is also required for catalysis. The magnesium ion possibly acts as an electrostatic shield. The enzyme pyruvate kinase, described earlier, and shown in Figure 1, requires both magnesium and potassium ions for maximal activity. 

FIGURE 17.6 Allosteric effects in phosphofructokinase. At high [ATP], phosphofructokinase behaves cooperatively, and the plot of enzyme activity versus [fructose-6-phosphate] is sigmoidal. High [ATP] thus inhibits PFK, decreasing the enzyme s affinity for fructose-6-phosphate. 

Phosphofmctokinase is also activated by fructose-2,6-bisphosphate, which is formed from fructose-1-phosphate by a second, separate phosphofructokinase enzyme—phosphofructokinase II (as shown in Figure 9-2). The activity of PFKII is itself regulated by hormone action. 

Adipose tissue obtained from diabetic animals shows a decrease of the incorporation of labeled amino acid into protein and in the activity of a number of enzymes hexokinase II, glucose-6-P04 dehydrogenase, phosphofructokinase and pyruvate kinase. Amino acid incorporation and enzyme activities return to normal after injection of insulin. The effect of insulin is, at least in the case of hexokinase II and glucose-6-PO4 dehydrogenase, impaired by the injection of actinomycin D indicating that insulin stimulates de novo protein synthesis by triggering the biosynthesis of new messenger RNA. 

Until now, we have mainly considered extracellular hydrogen ion concentration. The usefulness to the organism of a stable extracellular fluid is so that intracellular biochemical processes occur at constant hydrogen ion concentration. Enzymic activity is extremely sensitive to small changes in pH for example the enzyme phosphofructokinase increases in activity almost 20-fold if the pH changes from 7.1 to 7.2. 

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.

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