Regulation of glycolysis Glycolysis is regulated at 3 steps which are irreversible. These reactions are catalyzed by following key enzymes : (1) Hexokinase and glucokinase, (2) Phosphofructokinase I, and (3) Pyruvate kinase.Hexokinase and glucokinaseThese enzymes catalyze the first step of glycolysis, i.e., Glucose --> Glucose-6-phosphate. Glucokinase is found in liver, Whereas hexokinase is found in all tissues. Kinetic propeies of these two are different.Hexokinase has low Km, i.e., high affinity for glucose, low Vmax, and is subjected to feedback inhibition by the reaction product, glucose-6-phosphate. Hexokinase is found in most of the tissue except liver and comes into play when blood glucose is low. It is not affected by feeding or insulin. Hexokinase is not specific for glucose metabolism, it is also involved in metabolism of fructose and galactose.Glucokinase, on the other hand, is specific for glucose. It has high Km (i.e., low affinity for glucose), high Vmax and unlike hexokinase, it is not inhibited by glucose-6-phosphate. As it has low affinity for glucose (high km), it comes into play only when intracellular glucose concentration is high. It is induced by feeding and insulin. Glucagon inhibits glucokinase.Function of hexokinase is to provide glucose-6-phosphate at a constant rate, according the needs of cells, i.e., function of hexokinase is to provide constant glucose utilization by all tissues of body even when blood sugar is low. Function of glucokinase in the liver is to remove glucose from blood after a meal, providing glucose-6phosphate in excess of requirement for glycolysis so that it can be used for glycogen synthesis and lipogenesis.Phosphofructokinase IPhosphofructokinase I is the major regulatory enzyme of glycolysis. It catalyzes the 3rd reaction of glycolysis, i.e., fructose-6-P Fructose 1,6 bis-P. This reaction is irreversible and is the "rate -limiting step" for glycolysis. It is also the "commeted step", meaning that once fructose 1,6 bisphophate is formed it must go for the glycolytic pathway only. So, most impoant control point for glycolysis is through regulation of phosphofructokinase I.Phosphofructokinase - I is allosterically activated by : Fructose-6-phosphate, fructose 2,6-bisphophate, AMP, ADP, K+ and phosphate. It is allosterically inhibited by : ATP, citrate, Ca+2, Mg+2, and low pH. Phosphofructokinase is an inducible enzyme that increases its synthesis in response to insulin and decreases in response to glucagon.Fructose 2,6-bisphosphate (F-2,6-BP) is the most impoant allosteric modulator (activator) of phosphofructokinase-I. Fructose 2,6-bisphosphate is synthesized as a side product of glycolysis. A bifunctional enzyme named PFK-2/Fructose 2,6 bisphosphatase is responsible for regulating the level of fructose 2,6 bisphosphate in the liver. Phosphofuctokinase-2 (PFK-2) activity of this bifunctional enzyme is responsible for synthesis of F-2,6-BP from fructose-6-phosphate and fructose 2,6 bisphosphatase activity is responsible for hydrolysis of F-2,6-BP back to fructose-6-phosphate.