Adenosine Triphosphate (ATP) Regeneration

In bioengineering and life sciences, adenosine triphosphate (ATP) regeneration is a crucial aspect of designing cell-free systems, metabolic engineering, biosensors, and biocatalytic processes. Since ATP is a primary energy currency in biological systems, maintaining its availability through regeneration systems is vital for sustained biochemical reactions. Several enzymes are commonly employed to regenerate ATP from ADP or AMP using various phosphate donors.

1. Creatine Kinase (CK)

• Reaction:
  Creatine phosphate + ADP → Creatine + ATP

• Use:
  Popular in cell-free protein synthesis (CFPS) systems due to high efficiency and rapid ATP generation.

• Advantage: Fast and reversible, good for short-term applications.

2. Polyphosphate Kinase (PPK)

• Reaction:
  Polyphosphate (PolyP) + ADP → PolyP(n-1) + ATP

• Use:
  Utilized in synthetic biology and biocatalysis, especially when cost-effective and non-ATP phosphate donors are needed.

• Advantage: PolyP is inexpensive and stable; PPK enables long-term ATP supply.

3. Pyruvate Kinase (PK)

• Reaction:
  Phosphoenolpyruvate (PEP) + ADP → Pyruvate + ATP

• Use:
  Widely used in in vitro transcription/translation systems and enzymatic assays.

• Advantage: High yield of ATP; well-characterized enzyme.

4. Acetate Kinase (Ack)

• Reaction:
  Acetyl phosphate + ADP → Acetate + ATP

• Use:
  Alternative to CK in cell-free systems where acetyl phosphate is used as an energy source.

• Advantage: Acetyl phosphate is stable and inexpensive.

5. Succinate Thiokinase (Succinyl-CoA Synthetase)

• Reaction:
  Succinyl-CoA + ADP (or GDP) + Pi → Succinate + ATP (or GTP) + CoA

• Use:
  Used in engineered TCA cycle mimetics or synthetic metabolic pathways.

6. Arginine Kinase

• Reaction:
  Phosphoarginine + ADP → Arginine + ATP

• Use:
  Sometimes used in invertebrate models or specific biochemical assays.

Engineering Considerations

• Stability: Some phosphate donors degrade or are unstable (e.g., PEP), requiring enzyme and substrate stabilization.

• Cost: PolyP and acetyl phosphate are much cheaper than PEP or creatine phosphate, making them attractive for industrial applications.

• Compatibility: Enzymes must be compatible with the system (e.g., pH, temperature, cofactors).

• Byproduct Management: Accumulation of byproducts (like pyruvate) may inhibit reactions or require removal.

Applications in Bioengineering

• Cell-Free Protein Synthesis (CFPS): ATP regeneration systems are critical for sustaining translation.

• Biocatalysis: Enzyme cascades often require continuous ATP supply for ligase or synthetase activity.

• Synthetic Biology: In artificial cells or minimal systems, ATP regeneration is key for mimicking metabolic cycles.

• Biosensors: Enzymatic ATP regeneration can be used to amplify signals.