While most steps in gluconeogenesis are the reverse of those found inglycolysis, three regulated and strongly exergonic reactions are replaced with more kinetically favorable reactions. Hexokinase/glucokinase, phosphofructokinase, and pyruvate kinase enzymes of glycolysis are replaced with glucose-6- phosphatase, fructose-1,6-bisphosphatase, and PEP carboxykinase. This system of reciprocal control allow glycolysis and gluconeogenesis to inhibit each other and prevent the formation of afutile cycle.
The majority of the enzymes responsible for gluconeogenesis are found in the cytoplasm; the exceptions are mitochondrial pyruvate carboxylase, and, in animals, phosphoenol-pyruvate carboxykinase. The latter exists as an isozyme located in both the mitochondrion and the cytosol. As there is no known mechanism to transport phosphoenolpyruvate from the mitochondrion into the cytosol, the cytosolic enzyme is believed to be the isozyme important for gluconeogenesis. The rate of gluconeogenesis is ultimately controlled by the action of a key enzyme, fructose-1,6-bisphosphatase, which is also regulated through signal ransduction bycAMP and its phosphorylation.
Most factors that regulate the activity of the gluconeogenesis pathway do so by inhibiting the activity or expression of key enzymes. However, both acetyl CoA and citrate activate gluconeogenesis enzymes (pyruvate carboxylase and fructose-1,6-bisphosphatase, respectively). Due to the reciprocal control of the cycle, acetyl-CoA and citrate also have inhibitory roles in the activity of pyruvate kinase.