After glycolysis, the charged pyruvate molecules enter the mitochondria via active transport and undergo three enzymatic reactions. These reactions ensure that pyruvate can enter the next metabolic pathway so that energy stored in the pyruvate molecules can be harnessed by the cells.

First, the enzyme pyruvate dehydrogenase removes the carboxyl group from pyruvate and releases it as carbon dioxide. The stripped molecule is then oxidized and releases electrons, which are then picked up by NAD+ to produce NADH, forming acetate.

Finally, coenzyme A—a sulfur-containing compound derived from a B vitamin—attaches to the acetate via its sulfur atom to create acetyl coenzyme A, or acetyl CoA. Acetyl CoA then moves into the citric acid cycle where it will be further oxidized.