Phosphofructokinase catalytic activity

Phosphofructokinase has four identical subunits. To explore how sigmoidal kinetics can arise in such an enzyme, consider an enzyme that has just two such subunits, each with its own catalytically active site. We can schematize the binding of substrate to the enzyme as follows ... 

Figure 7.17. Effects of pH on binding of phosphofructokinase (PFK) to myofibrils (open symbols dashed line) and interacting effects of pH and temperature on the self-assembly of PFK, as indexed by residual PFK activity (filled symbols solid lines). Binding of PFK to myofibrils was measured using PFK-con-taining supernatants and myofibrillar preparations from white locomotory muscle of the teleost Paralabrax nebulifer (Roberts et al., 1998). PFK self-assembly was studied using purified enzyme from a mammal (Spermophilus beecheyi) (Hand and Somero, 1983). The percent residual PFK activity after incubation for 60 min under different combinations of pH and temperature provides an estimate of the fraction of PFK that remains as catalytically active tetramers or aggregates of tetramers. (Figure from Somero, 1997.)...

Most or all ATP-requiring enzymes use ATP in the form of the Mg-ATP complex. ATP chelates the magnesium ion, which is a divalent metal ion. The glycolytic pathway features a number of Mg-requiring enzymes. One of these enzymes is phosphofructokinase. The Mg requirement for this enzyme is illustrated by the data in Figure 10-50- Ihe study involved phosphofructokinase purified from rabbit muscle. Each point in Figure 10-50 represents the catalytic activity of the enzyme that was expressed during incubation in separate test tubes. All of the test tubes... 

Fructose 1,6-bisphosphatase is inhibited by AMP—a compound that activates phosphofructokinase Elevated AMP thus stimulates path ways that oxidize nutrients to provide energy for the cell. [Note ATP and NADH, produced in large quantities during fasts by catalytic path ways, such as fatty acid oxidation, are required for gluconeogenesis.]... 

Figure 9.9 shows the crystal structure of two of the subunits of phosphofructokinase from B. stearothermophilus. In the complete enzyme, the subunits are disposed symmetrically about three mutually perpendicular axes. Each of these axes is a twofold symmetry axis, which means that rotating the entire structure by half of a full circle (180°) around the symmetry axis results in an identical structure. This rotation is shown diagramatically in figure 9.10. Because ADP is a product of the enzymatic reaction as well as an allosteric activator, it binds at both the catalytic and allosteric sites (see figs. 9.9 and 9.10). The catalytic site for fructose-6-phosphate in each subunit is at the interface of the subunit with one of its neighbors, and the allosteric site is at the interface with a different neighbor. 

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