Though many of the steps of gluconeogenesis are the simple reversal of steps of glycolysis, there are three important steps in glycolysis that are replaced by four different steps in gluconeogenesis. Each of these steps is very energetically favored for glycolysis, so the gluconeogenesis pathway could not effectively reverse them. Instead, in two of the three steps, gluconeogenesis reactions omit regeneration of the ATP used in glycolysis, simply hydrolyzing the phosphate instead. This allows the reaction to proceed by "saving" the energy that would otherwise be put into regenerating ATP. The third glycolysis step that is bypassed is overcome by a set of two "sidestep" reactions in which ATP and GTP energy is expended to generate phosphoenolpyruvate, a high-energy intermediate, .

Figure 16.3 schematically illustrates the differences between glycolysis and gluconeogenesis. Six of the seven reactions common to the two pathways are not shown. In each case, the name of the gluconeogenesis enzyme that differs from the corresponding glycolysis enzyme is given. The reactions unique to gluconeogenesis are the following:

1. Pyruvate + CO2 + H2O + ATP <=> Oxaloacetate + ADP + Pi + 2H⁺ (= -2.1 kJ/mol).
This reaction is catalyzed by pyruvate carboxylase. Unlike the other reactions of glycolysis and gluconeogenesis, this reaction occurs in the mitochondrial matrix.

2. Oxaloacetate + GTP <=> Phosphoenolpyruvate + CO2 + GDP (=+2.9 kJ/mol)
This reaction is catalyzed by phosphoenolpyruvate carboxykinase - PEPCK.

3. Fructose-1,6-bisphosphate + H2O <=> Fructose-6-phosphate + Pi (=-16.3 kJ/mol)
This reaction is catalyzed by fructose 1,6 bisphosphatase.

4. Glucose-6-phosphate + H2O <=> Glucose + Pi ( = -12.1 kJ/mol)
This reaction is catalyzed by glucose-6-phosphatase.

In all the other reactions, gluconeogenesis proceeds simply by reversing the corresponding reaction of glycolysis. The entire pathway is summarized in Table 16.1. Note that the overall for gluconeogenesis is negative (-47.6 kJ/mol). Glycolysis too has an overall negative (-73.3 kJ/mol). The difference is that glycolysis accomplishes a negative while yielding reducing equivalents (2 NADH) and 2 net ATP, but the biosynthetic gluconeogenesis pathway requires use of 4 ATP and 2 GTP to achieve its overall negative .