Glycolysis is the only metabolic pathway common to nearly all living organisms, suggesting great antiquity; it may have originated with the first prokaryotes, 3.5 billion years ago or more.
The first step in glycolysis is phosphorylation of glucose by hexokinase. This reaction consumes 1 ATP molecule, but the energy is well spent: although the cell membrane is freely permeable to glucose, it is impermeable to glucose 6-phosphate. Glucose 6-phosphate is then rearranged into fructose 6-phosphate by phosphoglucose isomerase[?]. (Fructose can also enter the glycolytic pathway[?] at this point.)
Phosphofructokinase[?] then consumes 1 ATP to form fructose 1,6-biphosphate. The energy expenditure in this step is justified in 2 ways: the glycolytic process (up to this step) is now irreversible, and the energy supplied to the molecule allows the ring to be split by aldolase into 2 molecules, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. (Triosephosphate isomerase converts the molecule of dihydroxyacetone phosphate into a molecule of glyceraldehyde 3-phosphate.) Each molecule of glyceraldehyde 3-phosphate is then oxidized by a molecule of NAD+ in the presence of glyceraldehyde 3-phosphate dehydrogenase, forming 1,3-biphosphoglycerate.
In the next step, phosphoglycerate kinase forms a molecule of ATP while forming 3-phosphoglycerate. At this step glycolysis has reached the break-even point: 2 molecules of ATP were consumed, and 2 new molecules have been synthesized. This step, one of the two substrate-level phosphorylation steps, requires ADP; thus, when the cell has plenty of ATP (and little ADP) this reaction does not occur. Because ATP decays relatively quickly when it is not metabolized, this is an important regulatory point in the glycolytic pathway. Phosphoglyceromutase then forms 2-phosphoglycerate; enolate then forms phosphoenolpyruvate; and another substrate-level phosphorylation then forms a molecule of pyruvate and a molecule of ATP. This serves as an additional regulatory step.
After the formation of fructose 1,6 biphosphate, many of the reactions are energetically unfavorable. The only reactions that are favorable are the 2 substrate-level phosphoylation steps that result in the formation of ATP. These two reactions pull the glycolytic pathway to completion.
So, for prokaryotes, the metabolism of 1 molecule of glucose has a net yield of 2 molecules of ATP. Eukaryotes, which have mitochondria, produce an additional 34 molecules of ATP for each glucose molecule.