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The Biochemical Impact of Extracting an Embedded Adenylate Kinase Domain Using Circular Permutation.

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Circularly permuting adenylate kinases (AKs) maintains cellular activity and substrate binding. This topological change alters enzyme activity and folding dynamics, offering insights into enzyme engineering.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Adenylate kinases (AKs) possess distinct AMP-binding and lid domains within their polypeptide structure.
  • Circular permutation can alter protein topology and stability.

Purpose of the Study:

  • To investigate the impact of circular permutation on the activity, stability, and folding dynamics of mesophilic and thermophilic adenylate kinases.
  • To understand how topological restructuring affects enzyme function and substrate binding.

Main Methods:

  • Circular permutation of mesophilic and thermophilic AKs.
  • Assays for enzyme activity and substrate binding at various temperatures.
  • Thermal denaturation studies (melting temperature, unfolding transitions).
  • Proteolytic digestion and mass spectrometry to analyze protein stability and identify termini.

Main Results:

  • Permuted AKs retained cellular activity and substrate-binding characteristics.
  • Permutation decreased activity at physiological temperatures but increased thermophilic AK activity at lower temperatures (>30 °C below Tm).
  • Permuted AKs showed reduced thermostability and multiphasic unfolding compared to native AKs.
  • Proteolytic digestion indicated altered stability, with new termini in the AMP-binding domain contributing to increased proteolysis sensitivity.

Conclusions:

  • Circular permutation can modulate enzyme activity and folding dynamics by altering contact order.
  • Topological restructuring offers a strategy for tuning enzyme properties.
  • Understanding these changes provides insights into the relationship between protein structure, stability, and function.