Although glycolysis will ultimately produce 4 ATP, there is an initial requirement of 2 ATP for it to begin. The conversion of glucose to glucose-6-phosphate and the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate both require ATP.  

The first and third steps of glycolysis are both energetically unfavorable. This means they will require an input of energy in order to continue forward. Per glucose molecule, 1 ATP is required for each of these steps. Therefore, a total of 2 ATP is needed during the energy investment phase of glycolysis.  

In the glycolytic pathway, 2 molecules of ATP must be used. The purpose of these molecules is to phosphorylate 2 intermediates in the pathway:

1. Glucose must be phosphorylated to glucose-6-phosphate.

2. Fructose-6-phosphate must be phosphorylated to fructose-1,6-bisphosphate.

Hexokinase and glucokinase are two enzymes that serve similar roles but have different characteristics. Hexokinase is found in all tissues, is inhibited by glucose 6 phosphate, and is not induced by insulin. It has a physiologic role of providing cells with a basal level of glucose 6 phosphate necessary for energy production.

The reaction turning glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate is shown below

This step of glycolysis does not hydrolyze or generate ATP, even though a phosphate group was added onto the glyceraldehyde-3-phosphate. The energy released when  is reduced to , sometimes referred to as the energy of oxidation (of glyceraldehyde-3-phosphate).

Fructose can be directly transformed into fructose-6-phosphate by hexokinase.

Glycolysis, as the name suggests, is the process of lysing glucose into pyruvate. Since glucose is a six-carbon molecule and pyruvate is a three-carbon molecule, two molecules of pyruvate are produced for each molecule of glucose that enters glycolysis. Glycolysis occurs in the cytoplasm of the cell, and does not require oxygen. The net energy production is two ATP per glucose.

After glucose is converted into glucose-6-phosphate by hexokinase, glucose-6-phosphate is converted into fructose-6-phosphate. This reaction is catalyzed by phosphoglucose isomerase.

The sixth step of glycolysis involves the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH moves a hydrogen onto the electron acceptor  An NADH is created for each molecule of G3P formed. A phosphate group from inorganic phosphate instead of ATP replaces the hydrogen group that was taken from G3P. This creates the molecule 1,3-bisphosphoglycerate. Remember that glucose is a six-carbon sugar, and that both G3P and 1,3-bisphosphoglycerate are three-carbon sugars. Thus there are two of each of these three-carbon sugars yielded for each glucose that enters glycolysis.

The seventh reaction of glycolysis is the conversion of 1,3-bisphosphoglycerate into 3-phosphoglycerate. The phosphate group is transferred from 1,3-bisphosphoglycerate onto ADP, yielding ATP. The conversion is catalyzed by the enzyme phosphoglycerate kinase.