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Related Concept Videos

Protein Dynamics in Living Cells01:19

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
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Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
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The theory of catalytically perfect enzymes was first proposed by W.J. Albery and J. R. Knowles in 1976. These enzymes catalyze biochemical reactions at high-speed. Their catalytic efficiency values range from 108-109 M-1s-1. These enzymes are also called 'diffusion-controlled' as the only rate-limiting step in the catalysis is that of the substrate diffusion into the active site. Examples include triose phosphate isomerase, fumarase, and superoxide dismutase.
 
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Proteins show rotational as well as lateral diffusion across the membrane. The lateral diffusion of proteins was confirmed through the cell fusion experiment where mouse and human cells were fused, resulting in hybrid cells. When the human and mouse cells fused, the specific membrane proteins on human and mouse cells were marked with the red and green-fluorescent markers, respectively. Initially, the red and green fluorescence was located on the respective hemisphere of the cell. As time...
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Updated: Aug 5, 2025

Assaying Protein Kinase Activity with Radiolabeled ATP
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Kinetics of diffusion-influenced multisite phosphorylation with enzyme reactivation.

Irina V Gopich1, Attila Szabo1

  • 1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20892, USA.

Biopolymers
|March 29, 2023
PubMed
Summary

Accounting for diffusion in multisite phosphorylation requires more than modifying rate constants. Introducing enzyme reactivation leads to negative rate constants and necessitates a non-Markovian theory for accurate kinetic modeling.

Keywords:
bistabilitydiffusion-limited reactionmemory kernelsphosphorylation

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Area of Science:

  • Biochemistry
  • Chemical Kinetics
  • Enzyme Kinetics

Background:

  • Multisite phosphorylation kinetics are complex and influenced by diffusion.
  • Conventional rate equations may not fully capture diffusion effects.
  • Enzyme inactivation and reactivation add further complexity.

Purpose of the Study:

  • To extend diffusion-modified kinetic models to include enzyme reactivation.
  • To develop a more accurate theoretical framework for short-time kinetics.
  • To investigate the impact of enzyme reactivation on phosphorylation cycles.

Main Methods:

  • Modification of conventional rate equations to include diffusion.
  • Introduction of new transitions and enzyme reactivation steps.
  • Development of a non-Markovian theory with memory kernels.
  • Application to double phosphorylation and phosphorylation-dephosphorylation cycles.

Main Results:

  • A diffusion-modified kinetic scheme with a negative rate constant emerged due to enzyme reactivation.
  • Non-Markovian effects are significant at short times.
  • The non-Markovian theory accurately describes short-time kinetics.
  • Loss of bistability in a phosphorylation-dephosphorylation cycle was reproduced with decreasing enzyme reactivation time.

Conclusions:

  • Enzyme reactivation significantly alters diffusion-influenced phosphorylation kinetics.
  • A non-Markovian approach is essential for accurate modeling at short timescales.
  • The developed theory provides a more robust framework for understanding complex enzymatic reactions.