ATP SYNTHESIS BY SUBSTRATE LEVEL PHOSPHOYRYATION

E. coli generates cell energy in the form of ATP to fuel a variety of cellular processes needed for cell biosynthesis, reproduction and maintenance. This is accomplished by one of two mechanisms termed Substrate-Level Phosphorylation (SLP) and Respiration-Linked Phosphorylation (RLP). Formation of ATP by SLP occurs when sugars like glucose are broken down into smaller molecular weight phosphorylated intermediates which in turn are used to synthesize ATP from ADP.

KEY CONCEPTS

  • Substrate level phosphorylation (SLP) is one of the two ways cells generates ATP.
  • During the SLP process, a phosphate moiety is transferred from a suitable donor substrate to ADP to form ATP. The donor molecule must have sufficient energy to support the reaction.
  • SLP reactions are catalyzed by kinase enzymes and E. coli contains at least four distinct enzymes.
  • Two of the kinase enzymes in E. coli participate in the glycolysispathway. The third kinase is made when cells produce acetyl-phosphate during fermentation conditions and the fourth enzyme participates in the TCA cycle by reversing its ATP-dependent reaction.
  • SLP can occur under aerobic and/or anaerobic cell growth conditions. However, under anaerobic, fermentative growth conditions, SPL generates the vast majority of ATP.

GENERAL BACKGROUND ON ATP SYNTHESIS BY SLP

At least four enzymes generate ATP by substrate-level phosphorylation (SLP) in E. coli. They share the ability to capture the phosphate anhydride bond energy from a suitable cellular intermediate and transfer it to ADP, thereby producing ATP.

Each reaction is catalyzed by substrate specific kinase enzyme whereby two are members of the glycolysis pathway. The third kinase enzyme, acetate kinase is made when excess acetyl-phosphate is present in the cell. Finally, succinyl-CoA synthetase is the only enzyme in the TCA cycle that generates ATP by SPL.

All four enzymes operate under many aerobic and anaerobic cell growth conditions where the newly formed ATP is used to fuel many energy consuming biosynthetic as well as cell maintenance reactions.

SLP provides a steady supply of ATP when E. coli is growing on glucose and on sugars that are easily converted to it. Under anaerobic fermentation conditions, almost all ATP is made by this process. However, when cells respire under aerobic or anaerobic conditions, ATP is also made by the alternative process called respiratory linked phosphorylation (RLP).

Aerobic Respiration

Anaerobic Respiration

The Enzymes

1,3 bisphosphoglycerate + ADP ↔ 3-phosphoglycerate + ATP

The phosphorylated 3-carbon substrate used in this reaction is generated during the oxidation of glucose by the glycolysis pathway. The reaction is reversible.

phosphoenolpyruvate + ADP ↔ pyruvate + ATP

Phosphoenolpyruvate (PEP) is also an intermediate in of the glycolysis pathway. E. coli employs two isoenzymes, PykA and PykF to catalyze the same reaction. Transcription of the pykA gene is stimulated under anaerobic conditions by the Fnrregulatory protein. The pyruvate kinase reaction is reversible.

acetyl-phosphate + ADP ↔ acetate + ATP

Acetyl-phosphate is produced from acetyl-CoA by phosphate acetyltransferase during fermentation conditions. Acetyl-CoA can be generated by several pathways including the oxidation of pyruvate following the glycolysis pathway when E. coligrows on sugars or by the fatty acids β-oxidation pathway.

Other Bacteria Produce Alternative Kinase Enzymes

Besides the enzymes made by E. coli, certain bacteria employ alternative kinase enzymes that couple to SLP. For example, propionate kinase and butyrate kinase harvest the bond energy stored in their respective substrates, propionyl-phosphate and butyryl-phosphate.

Propionate kinase is present in certain bacteria (e.g. Propionibacterium) that ferment lactate and produce propionic acid as an end product.

propionyl-phosphate + ADP ↔ propionate + ATP

Butyrate kinase is present in some other anaerobic bacteria (e.g. Clostridium butyricum) that ferment sugars and produce butyrate as an end product.

butyryl-phosphate + ADP ↔ butyrate + ATP

Only Some Phosphate-Containing Compounds are Suitable Donors for SLP

The hydrolysis of one molecule of ATP to ADP plus inorganic phosphate releases approximately 32 kJ of energy per mole under standard conditions. Thus, the kinase-dependent formation of ATP from ADP and the donor molecule requires an equal or greater amount of energy than the ATP hydrolysis value. Phosphate-containing molecules with lower energy content cannot be used to drive ATP synthesis by SLP reactions.

The following Table lists the hydrolysis values for some commonly occurring carbon-phosphate molecules :

Table 1 – Free Energy Released During Hydrolysis

Reaction
ΔG°
phosphoenolpyruvate → pyruvate + Pi -52 kJ/mole
1,3-bisphosphyglycerate → 3-phosphyglycerate + Pi -52 kJ/mole
glucose-6-phosphate → glucose + Pi -14 kJ/mole
fructose-6-phosphate → fructose+ Pi -6 kJ/mole
acetyl-phosphate → butyrate + Pi -45 kJ/mole
propionyl-phosphate → butyrate + Pi -36 kJ/mole
butyryl-phosphate → butyrate + Pi -36 kJ/mole
succinyl-phosphate → butyrate + Pi -35 kJ/mole
ATP → ADP + Pi -32 kJ/mole
ADP → AMP + Pi -32 kJ/mole
AMP → adenosine + Pi -14 kJ/mole
ATP → AMP + PiPi -42 kJ/mole
PiPi → 2 Pi -22 kJ/mole

ΔG° – Free energy released per mole of substrate (KJ/mole) hydrolyzed under standard conditions

Please note that there are other kinase enzymes that do not generate ATP. Rather, they consume ATP to fuel various biosynthetic reactions. Examples of these non-SLP kinase enzymes include phosphotransferase, (nucleotidytransferase, phosptidyltranferase, diphosphotransferase, glycotransferase, and protein kinase.

Summary

  • Cells require large amounts of ATP to fuel the many types of energy consuming reactions.
  • ATP synthesis occurs by two distinct mechanisms named SLP and RLP.
  • The strategy used by a cell to make ATP by SLP involves transfer of a phosphate from an appropriate substrate to ADP.
  • Not all phosphorylated carbon compounds are suitable substrates for SLP.

Credits:

Authored by Robert Gunsalus and Imke Schröder
©The Escherichia coli Student Portal

This project acknowledges support from:
NIH Grant Award GM077678 to SRI, International
Peter Karp and coworkers at EcoCyc.org
The UCLA Department of MIMG