Enzyme kinetics studies have revealed that allosteric enzymes exhibit sigmoidal kinetics rather than the hyperbolic Michaelis-Menten kinetics observed in non-allosteric enzymes. This difference arises from cooperative binding at multiple active sites.

When substrate binds to one active site, conformational changes occur that affect the affinity of remaining sites. In positive cooperativity, initial substrate binding increases affinity at other sites; in negative cooperativity, it decreases affinity. ATP-dependent enzymes often display feedback inhibition, where the final product of a metabolic pathway inhibits the first enzyme in that pathway.

Research has shown that phosphofructokinase (PFK), a key regulatory enzyme in glycolysis, is allosterically inhibited by ATP and citrate while being activated by AMP and ADP. This regulation ensures that glycolysis proceeds only when cellular energy demands are high.

Studies using purified PFK in vitro demonstrated that at physiological pH (7.4), increasing ATP concentrations from 0.1 mM to 5 mM decreased enzyme activity by 75%, while increasing AMP from 0 to 0.5 mM restored activity to 90% of maximum. These findings suggest that the ATP:AMP ratio serves as a cellular energy sensor.

[Figure 1: Graph showing PFK activity vs. ATP concentration at various AMP levels]