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 glycolysis pathway. 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.


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.


  1. Phosphoglycerate kinase (Pgk)       
  2. Pyruvate kinase (PykA and PykF)       
  3. Acetate kinase (Ack)       
  4. Succinyl-CoA synthetase (SucCD)       

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.

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

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


  • 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.



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