4.2.8 Substrate Level Phosphorylation

Introduction

Substrate level phosphorylation is one of the two ways by which ATP is produced during cellular respiration. Unlike oxidative phosphorylation, which uses a proton gradient and the electron transport chain, substrate level phosphorylation involves the direct transfer of a phosphate group from an organic substrate molecule to ADP, forming ATP. It occurs during glycolysis and the Krebs cycle.

Definition

Substrate Level Phosphorylation: When phosphate is enzymatically transferred from an organic substrate molecule, it is called substrate level phosphorylation.

In simple words: Substrate level phosphorylation is the direct transfer of a phosphate group from a substrate molecule to ADP to make ATP.

Core Concept

• The prime objective of cellular respiration is to generate ATPs.
• There are two ways to do this during aerobic respiration:
• As far as substrate level phosphorylation is concerned, the addition of inorganic phosphate to any organic molecule is called phosphorylation.
• But when phosphate is enzymatically transferred from an organic substrate molecule, it is called substrate level phosphorylation.
• However, it accounts for only a small percentage of the ATP that a cell generates.
• It occurs at three occasions during aerobic respiration:

Comparison with Oxidative Phosphorylation

Detailed Explanation

Substrate Level Phosphorylation in Glycolysis

Glycolysis occurs in the cytoplasm. It involves two substrate level phosphorylation reactions:

Reaction 7: Formation of ATP from 1,3-Bisphosphoglycerate

Reaction: 1,3-Bisphosphoglycerate is converted into 3-phosphoglycerate by transferring its phosphate group to ADP, and a molecule of ATP is formed.

Enzyme: Phosphoglycerate kinase

• 1,3-Bisphosphoglycerate has a high-energy phosphate bond.
• The phosphate group is directly transferred to ADP.
• ATP is formed.
• 3-Phosphoglycerate is produced.
• This reaction occurs twice per glucose molecule (because glucose is split into two G3P molecules).

Equation: 1,3-Bisphosphoglycerate + ADP → 3-Phosphoglycerate + ATP

Reaction 10: Formation of ATP from Phosphoenol Pyruvate (PEP)

Reaction: A molecule of ATP is produced by substrate level phosphorylation when phosphoenol pyruvate loses its phosphate group, which is taken up by ADP to form ATP. The phosphoenol pyruvate is finally converted into pyruvate.

Enzyme: Pyruvate kinase

• PEP has a high-energy phosphate bond.
• The phosphate group is directly transferred to ADP.
• ATP is formed.
• Pyruvate is produced.
• This reaction occurs twice per glucose molecule.

Equation: Phosphoenol pyruvate (PEP) + ADP → Pyruvate + ATP

ATP Yield from Glycolysis

Important Note: There are four ATPs produced by substrate level phosphorylation during glycolysis, but two of them are consumed in the preparatory phase, so the net product by substrate level phosphorylation is 2 ATP.

Substrate Level Phosphorylation in Krebs Cycle

The Krebs cycle occurs in the mitochondrial matrix. It involves one substrate level phosphorylation reaction:

Reaction 6: Formation of ATP from Succinyl CoA

Reaction: Coenzyme A is removed from Succinyl CoA by the help of enzyme succinyl thiokinase to form succinate. The reaction releases sufficient energy which is used to combine GDP and Pi forming GTP. GTP reacts with ADP to form ATP while GTP is again converted into GDP. In this way a molecule of ATP is generated in this reaction.

Enzyme: Succinyl thiokinase

• Succinyl CoA has a high-energy thioester bond.
• The energy released is used to combine GDP and Pi to form GTP.
• GTP then transfers its phosphate group to ADP, forming ATP.
• Succinate is produced.
• This reaction occurs twice per glucose molecule (because the Krebs cycle turns twice).

Equation: Succinyl-CoA + GDP + Pi → Succinate + GTP + CoA GTP + ADP → ATP + GDP

ATP Yield from Krebs Cycle

Total ATP Yield from Substrate Level Phosphorylation

• Total ATP from aerobic respiration: 36–38 ATP
• ATP from substrate level phosphorylation: 4 ATP
• Percentage: ~11%
• ATP from oxidative phosphorylation: 32–34 ATP
• Percentage: ~89%

Substrate Level Phosphorylation vs. Oxidative Phosphorylation

Importance of Substrate Level Phosphorylation

• It does not require oxygen.
• It occurs in both aerobic and anaerobic respiration.
• It provides ATP when oxygen is limited.

• It is faster than oxidative phosphorylation.
• It provides quick energy for cells.

• It is essential for the net production of ATP in glycolysis.
• It ensures glycolysis can continue.

• It occurs in both prokaryotes and eukaryotes.
• It is a universal mechanism for ATP production.

5. Accounts for a small but important fraction of ATP: - Although it produces only 4 ATP, it is still essential.

Intermediate Level

Substrate Level Phosphorylation in Glycolysis

Note: Each reaction occurs twice per glucose molecule, so 4 ATP are produced. But 2 ATP are consumed in the preparatory phase, so the net is 2 ATP.

Substrate Level Phosphorylation in Krebs Cycle

Note: The Krebs cycle turns twice per glucose molecule, so 2 ATP are produced.

Total ATP Yield from Substrate Level Phosphorylation

Comparison with Oxidative Phosphorylation

Important Points

• 1. The prime objective of cellular respiration is to generate ATPs.
• 2. There are two ways to do this during aerobic respiration:
• - Chemiosmosis
• - Substrate level phosphorylation
• 3. As far as substrate level phosphorylation is concerned, you are already familiar that the addition of inorganic phosphate to any organic molecule is called phosphorylation.
• 4. But when phosphate is enzymatically transferred from an organic substrates molecule it is called substrate level phosphorylation.
• 5. However, it accounts for only a small percentage of the ATP that a cell generates.
• 6. It occurs at three occasions during aerobic respiration:
• - In glycolysis (when 1,3-bisphosphoglycerate is converted into 3-phosphoglycerate; 7th reaction)
• - In glycolysis (when phosphoenol pyruvate is converted into pyruvate; 10th reaction)
• - In Krebs cycle (when succinyl CoA is converted into succinate)
• 7. There are four ATPs produced by this mechanism during glycolysis but two of them are supposed to be consumed in the preparatory phase, so net product by substrate level phosphorylation is 2 ATP.
• 8. In the Krebs cycle, substrate level phosphorylation occurs when succinyl CoA is converted into succinate.
• 9. There are two molecules of ATP produced at this occasion.
• 10. Since ATP can be synthesized directly from the organic substrates of exergonic reactions (energy-releasing reactions, e.g., cellular respiration), therefore, it is said that substrate level phosphorylation couples the exergonic reactions with the synthesis of ATP.
• 11. These ATP are then used to drive endergonic reactions (energy-storing reactions, e.g., protein synthesis).
• 12. In this way, out of total 36 ATP which are produced during aerobic respiration in most of human cells, 4 ATP are the result of substrate level phosphorylation and remaining 32 ATP are produced by chemiosmosis through electron transport chain.

Remember

• - Substrate level phosphorylation = direct transfer of phosphate from an organic substrate to ADP
• - Location = cytoplasm and mitochondrial matrix
• - Occurs in = glycolysis and Krebs cycle
• - Glycolysis: 2 ATP (net)
• - Krebs cycle: 2 ATP
• - Total: 4 ATP
• - No oxygen required
• - Fast
• - Small percentage of total ATP (~11%)
• - Enzymes: Phosphoglycerate kinase, Pyruvate kinase, Succinyl thiokinase
• - Substrate level phosphorylation = 4 ATP
• - **Oxidative phosphorylation** = 32–34 ATP

Common Mistakes

• 1. Confusing substrate level phosphorylation with oxidative phosphorylation – Substrate level = direct transfer; Oxidative = via ETC and chemiosmosis.
• 2. Thinking substrate level phosphorylation requires oxygen – It does not require oxygen.
• 3. Forgetting the location – Substrate level phosphorylation occurs in the cytoplasm (glycolysis) and mitochondrial matrix (Krebs cycle).
• 4. Confusing the ATP yield – Substrate level = 4 ATP; Oxidative = 32–34 ATP.
• 5. Thinking substrate level phosphorylation occurs only in glycolysis – It also occurs in the Krebs cycle.
• 6. Forgetting the enzymes – Phosphoglycerate kinase, Pyruvate kinase, Succinyl thiokinase.
• 7. Thinking substrate level phosphorylation produces most of the ATP – It produces only a small percentage of the total ATP.
• 8. Confusing the reactions in glycolysis – Reactions 7 and 10 are substrate level phosphorylation.
• 9. Thinking GTP is not involved – GTP is an intermediate in the Krebs cycle reaction.
• 10. Forgetting the net ATP yield in glycolysis – Net ATP = 2 (4 produced – 2 consumed).
• 11. Confusing the total ATP yield – Total from aerobic respiration = 36–38 ATP; Substrate level = 4 ATP.
• 12. Thinking substrate level phosphorylation only occurs in prokaryotes – It occurs in both prokaryotes and eukaryotes.

Quick Revision

• The prime objective of cellular respiration is to generate ATPs.
• There are two ways to do this during aerobic respiration: chemiosmosis and substrate level phosphorylation, the former we have already discussed.
• As far as substrate level phosphorylation is concerned, you are already familiar that the addition of inorganic phosphate to any organic molecule is called phosphorylation but, when phosphate is enzymatically transferred from an organic substrates molecule it is called substrate level phosphorylation.
• However, it accounts for only a small percentage of the ATP that a cell generates.
• It occurs at three occasions during aerobic respiration.
• In glycolysis, substrate level phosphorylation occurs, when 1,3-bisphosphoglycerate is converted into 3-phosphoglycerate (7th reaction) and when phosphoenol pyruvate is converted into pyruvate (10th reaction).
• There are four ATPs produced by this mechanism during glycolysis but two of them are supposed to be consumed in preparatory phase so net product by substrate level phosphorylation is 2 ATP.
• In Krebs cycle, substrate level phosphorylation occurs when succinyl CoA is converted into succinate.
• There are two molecules of ATP produced at this occasion.
• Since, ATP can be synthesized directly from the organic substrates of exergonic reactions (energy releasing reactions e.g., cellular respiration), therefore, it is said that substrate level phosphorylation couples the exergonic reactions with the synthesis of ATP.
• These ATP are then used to drive endergonic reactions (energy storing reaction e.g., protein synthesis).
• In this way, out of total 36 ATP which are produced during aerobic respiration in most of human cells, 4 ATP are the result of substrate level phosphorylation and remaining 32 ATP are produced by chemiosmosis through electron transport chain.

60-Second Revision

• Substrate level phosphorylation = direct transfer of phosphate from an organic substrate to ADP
• Location = cytoplasm and mitochondrial matrix
• Occurs in = glycolysis and Krebs cycle
• Glycolysis: 2 ATP (net)
• Krebs cycle: 2 ATP
• Total: 4 ATP
• No oxygen required
• Fast
• Small percentage of total ATP (~11%)
• Enzymes: Phosphoglycerate kinase, Pyruvate kinase, Succinyl thiokinase
• Substrate level phosphorylation = 4 ATP
• **Oxidative phosphorylation** = 32–34 ATP
• Warning: Substrate level phosphorylation does not require oxygen. It occurs in the cytoplasm and mitochondrial matrix. It produces only 4 ATP, which is a small percentage of the total ATP. Oxidative phosphorylation produces 32–34 ATP. The enzymes are phosphoglycerate kinase, pyruvate kinase, and succinyl thiokinase. In glycolysis, 4 ATP are produced but 2 are consumed, so the net is 2 ATP. In the Krebs cycle, 2 ATP are produced. Substrate level phosphorylation couples exergonic reactions with ATP synthesis.