What Is Substrate Phosphorylation?

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  • Written By: E.A. Sanker
  • Edited By: John Allen
  • Last Modified Date: 12 October 2019
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Substrate phosphorylation, also called substrate-level phosphorylation, is a biochemical process by which cells make adenosine triphosphate (ATP) from adenosine diphosphate (ADP). This process occurs in the cytoplasm and is an important step in the metabolic pathway known as glycolysis. ATP is a cofactor, or coenzyme, which means that although not a protein itself, it is essential in driving reactions, transferring energy, and acting as a fuel source for the cell.

In order to produce ATP through substrate phosphorylation, an inorganic phosphate group must be transferred to ADP from a more energetic molecule. The phosphate group consists of four oxygen atoms bound to a central phosphorus atom and carries a negative charge. An enzyme mediates the reaction between ADP and the phosphate compound. The products of the reaction are ATP and another compound consisting of hydrogen, oxygen, and sometimes phosphorus. Taking the reaction as a whole, we can visualize ADP, with its two phosphate groups, being converted into ATP, a molecule with three phosphate groups, through the addition of one phosphate group from another molecule.

Substrate phosphorylation occurs twice during glycolysis, a multi-step metabolic pathway essential to living organisms. In glycolysis, the sugar glucose is converted into the organic acid pyruvate and ATP. This process is at the core of metabolism, allowing organisms to transform the sugar that they gain from nutrients into energy.


During the early substrate phosphorylation step in glycolysis, a phosphate group is transferred to ADP from a compound called 1,3-bisphosphoglycerate. The two substrates, ADP and 1,3-bisphosphoglycerate, bind to the enzyme phosphoglycerate kinase, which catalyzes the reaction. ATP and 3-phosphoglycerate are produced.

The final step of glycolysis also involves substrate phosphorylation. Phosphoenolpyruvate, a high-energy phosphate compound, transfers its phosphate group to ADP through the enzyme pyruvate kinase. The products are ATP and pyruvate, a molecule consisting of hydrogen and oxygen.

Substrate phosphorylation is regulated by external factors and may not always occur during glycolysis. When a cell has a large amount of ATP but little ADP, for example, the reaction may not proceed because there is not enough ADP to use. The presence of ATP itself can also inhibit the enzymes involved.

Hormones play a role in regulating glycolysis as well. Low blood glucose level, also known as low blood sugar, results in the production of glucagon. This hormone is produced in the pancreas and raises blood sugar. It inhibits the activity of pyruvate kinase in the final glycolysis step, preventing substrate phosphorylation from taking place.


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