The allosteric enzyme, phosphofructokinase, carries out the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate, the second ATP utilization in glycolysis. At this stage in glycolysis we have formed a high energy 'activated' compound at the cost of two ATPs. It is extremely important to note that the level of activity of phosphofructokinase is the primary regulator of the pace of glycolysis. Discussion of phosphofructokinase is a classic of biochemistry which has found its way in various ways onto the MCAT. Phosphofructokinase is inhibited by high levels of ATP, which lowers the affinity of the enzyme for fructose 6-phosphate. However, this inhibitory action is reversed by AMP, so the enzyme increases when the ATP/cAMP ratio (energy charge) is lowered. This is important. High cAMP turns on phosphofructokinase.

Furthermore, high citrate concentration, which indicates a high level of biosynthetic precursors, inhibits phosphofructokinase. What does that mean? Think for a moment. This is not only about inhibition by the products of glycolysis. High citrate concentrations cause a redirection of the fructose 6-phosphate towards biosynthesis instead of glycolytic breakdown.

Additionally, phosphofructokinase is stimulated by fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate is, in fact, the most potent regulator of glycolysis, produced as part of the second messenger system in response to the hormone glucagon. The effect of glucagon on glycolysis (and gluconeogenesis in reverse) is mediated first by cAMP and subsequently by Fructose 2,6-bisphosphate.