In order to think about how glycolysis is regulated, we will analyze data from t

Question

In order to think about how glycolysis is regulated, we will analyze data from the hyperthermophilic Archaeon, Pyrococcus furiosus. This organism grows optimally near 100 degree C: and can use maltose, cellobiose. starch, and proteins as energy sources. It is one of the few organisms that have tungsten-containing enzymes. In eukaryotes, phosphofructokinase (PFK) is the main site of regulation of glycolysis. ATP is an allosteric inhibitor of the PFK activity. ATP binding induces a conformational change in PFK to a T-state with lower affinity for fructose-6-P. Draw a [fructose-6-P] versus reaction velocity curve of PFK in the high versus low [ATP], and explain in two sentences or less why the curves differ. Be sure to label! AMP competes with ATP for binding at the allosteric site, but does not prevent fructose-6-P binding. With an arrow, indicate on your drawing above which curve best represents the activity of PFK when [AMP] is high.

Explanation / Answer

Phosphofructokinase-1 (PFK-1) is a glycolytic enzyme that catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate (F6P) to yield ADP and fructose-1,6-bisphosphate (FBP). Mg2+ is also important in this reaction (click here to see animation of reaction). Phosphofructokinase-2 (PFK-2) acts on the same substrates to yield ADP and fructose-2,6-bisphosphate (F2,6P). Click here to see the difference between FBP and F2,6P. PFK reaction is strongly exergonic (irreversible) under physiological conditions and hence is one of the glycolytic pathway's rate-determining steps. In most organisms/tissues, PFK is the glycolytic pathway's major flux-regulating enzyme; its activity is controlled by the concentrations of an unusually large number of metabolites including ATP, ADP, AMP, PEP and fructose-2,6-bisphosphate. PFK from B. stearothermophilus is a tetramer of identical 320-residue subunits. It is an allosteric enzyme that is described using the symmetry model of allosterism whereby there is a concerted transition from its high-activity R state to its low-activity T state. The X-ray structures of both R and T states of the enzyme have been reported.[1] The binding of one molecule of its substrate F6P, which binds to the R state enzyme with high affinity but to the T state enzyme with low affinity, causes PFK to take up the R state, which in turn, increases the binding affinity of the enzyme for additional F6P (a homotropic effect). Activators, such as ADP and AMP bind to so-called allosteric sites, binding sites distinct from the active site, where they likewise facilitate the formation of the R state and hence activate the enzyme (a heterotropic effect; ADP, being a product of the PFK reaction, also binds at the enzyme's active site). Similarly, inhibitors such as PEP bind to allosteric sites (which in the case of PFK overlaps the activating allosteric site) where they promote the formation of the T state, thereby inhibiting the enzyme.

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