The first step in glycolysis is the conversion of glucose to glucose-6-phosphate through the consumption on one ATP molecule. Glucose is reacted upon by the enzyme hexokinase to carry out this step. Kinases are a group of enzymes that add phosphate groups by removing them from an ATP. All of these other enzymes catalyze subsequent reactions in glycolysis.

Glyceraldehyde phosphate dehydrogenase (GAPDH) is used in the sixth reaction, where G3P is converted to 1,3-bisphosphoglycerate (1,3-BPG). A hydrogen is removed from G3P and added to , yielding NADH. Also, G3P has one phosphate group, while 1,3-BPG has two. The energy released as G3P is oxidized (causing subsequent reduction of ) is highly exergonic. This energy, sometimes referred to as the energy of oxidation, drives the addition of inorganic phosphate onto G3P, yielding the doubly-phosphorylated 1,3-BPG.

Dihydroxyacetone phosphate (DHAP) is converted to glyceradehyde-3-phosphate (G3P) by the enzyme triose phosphate isomerase. As the name suggests, this enzyme catalyzes the isomerization of a three-carbon sugar into another three-carbon sugar. Since the molecular formulas of DHAP and G3P are the same, we know that they are isomers of each other.

The balance between DHAP and G3P is extremely important in regulating overall cell metabolism. DHAP is a precursor to triglycerides, and is used in their synthesis, while G3P is an intermediate in glycolysis, an ATP-producing process. In order to favor the conversion of DHAP into G3P, and not the opposite, the cell must keep G3P levels low (Le Chatelier's Principle). Consider the following equilibrium: . This should make sense: if there is lots of ATP around in the cell, there is no need for glycolysis to proceed. Thus the equilibrium will be pushed to the left, increasing the concentration of DHAP in the cell. In humans, DHAP is converted into triglycerides, which get stored as fat. One way to shift this equilibrium to the right is to "create" an ATP need. This can be done by exercising. Exercise utilizes ATP and will thus pull the equilibrium to the right, removing DHAP (which was destined to be converted into fat) and facilitates its conversion into G3P to proceed with cellular respiration.

The tenth and final reaction of glycolysis involves the conversion of phosphoenolpyruvate (PEP) into pyruvate. This step is catalyzed by the enzyme pyruvate kinase. This kinase is going to remove a phosphate group from PEP and put it on ADP to yield ATP. Pyruvate, a three-carbon molecule, is the end product of glycolysis. It can be sent to the pyruvate dehydrogenase complex to be turned into acetyl-CoA, which enters the Krebs cycle. Alternatively, it can be reduced into lactate and/or ethanol (depending on the organism) to regenerate for glycolysis via anaerobic respiration.

Theninth reaction involves the conversion of 2-phosphoglycerate into phosphoenolpyruvate. The enzyme enolase, which produces a double bond by removing the hydroxyl group on 2-phosphoglycerate catalyzes this reaction. Note that the resulting molecule is an enol (double bond -ene, and alcohol - ol).

Isomerases catalyze the isomerization, or rearrangement of atoms within a molecule, of its substrate. Isomerases are seen in glycolysis inn the second step where glucose-6-phosphate is converted into fructose-6-phosphate by phosphoglucose isomerase. Glucose-6-phosphate is rearranged into fructose-6-phosphate such that the molecular formula is unchanged. Another isomerase is triose phosphate isomerase. It catalyzes the isomerization of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate.

Hexokinase is the first enzyme in the glycolytic pathway and it is responsible for the phosphorylation of glucose to glucose 6-phosphate. The other enzymes catalyze subsequent reactions in glycolysis.

Glyceraldehyde 3-phosphate dehydrogenase is the only enzyme in glycolysis that carries out a redox reaction. Glyceraldehyde 3-phosphate is oxidized to 1,3-bisphosphoglycerate while is reduced to .

The rate-limiting step of glycolysis is the conversion of glucose-6-phosphate to fructose-6-phosphate. This reaction is catalyzed by the enzyme phosphofructokinase. Hexokinase catalyzes the conversion of glucose to glucose-6-phosphate, pyruvate kinase converts Phosphoenolpyruvate to pyruvate, and lactate dehydrogenase converts pyruvate into lactose.

Phosphofructokinase catalyzes the third step in glycolysis transforming fructose 6-phosphate to fructose-1,6-bisphosphate. It is an irreversible step, and it is one of the major regulatory points of glycolysis. One way in which it controls the flow of glycolysis is that when there is a high level of ATP, PFK is inhibited. This is because the ultimate goal of glycolysis is to make ATP. Thus, if there is already a high level of ATP, glycolysis should slow down.