Keywords

Protein Kinase, Catalysis, Kinetics, Phosphorylation, Enzyme Mechanisms, PKA, Activation Loop, Metal Ions, QM/MM

Reference

DOI: 10.1021/cr000230w

Abstract

Detailed analysis of protein kinase catalysis and kinetics, primarily using PKA as a model. Covers substrate recognition, metal ion function, phosphoryl transfer, activation loop, and regulation. Offers insights into catalytic steps, binding mechanisms, and rate-limiting processes.

Notes

1. General Summary

  • Protein kinases exhibit poor phosphorylation of free amino acids, requiring flanking sequences (P-site, P-1, P-2, etc.).

  • Consensus sequences dictate specificity; distal recognition elements are often essential for efficient phosphorylation.

  • Bisubstrate kinetics: Order of ATP and substrate binding can vary; for large protein substrates, ATP access may be delayed or induced by conformational changes.

  • Focus on key residues:

    • Asp184: Mg2+ coordination, phosphate positioning, charge shielding.
    • Lys72: Stabilizes α/β phosphates.
    • Lys168: Interacts with γ phosphate, impacts binding.
    • Asp166: Potential substrate positioning (general base role debated).


    Figure: Some important sites (Click to enlarge)

2. Activation Loop: Gate or Not?

  • Unphosphorylated activation loops may occupy the active site (“door hypothesis”).
  • Phosphorylation reduces loop disorder (B factors), enabling substrate access.
  • However, mixed experimental support:
    • Enhances substrate binding for ERK2, cdk2.
    • No effect in PKA and v-Fps.
    • 2-4 order magnitude activation effect on catalysis when phosphorylated.
  • Not universal: PhK lacks phosphorylation site, but regulated through other structural shifts.

3. Phosphoryl Transfer Mechanism

  • Direct single-step phosphate displacement without intermediates (unlike phosphatases).
  • Dissociative transition state model favored:
    • 91.6% dissociative, 8.4% associative in PKA.
    • Bond-breaking dominates transition state.

4. Metal Ion Roles

  • Mg2+ essential for catalysis:
    • Mg1: Core cofactor.
    • Mg2: Observed in some structures under high [Mg2+]; modulates catalysis.
  • First metal (activating), second metal (complex role: sometimes inhibitory, sometimes enhancing velocity at low ATP).
  • Metal types affect activity:
    • Mn2+, Ca2+, Co2+, Zn2+, but Mg2+ is primary in vivo.
    • TPK: uniquely prefers Mn2+ over Mg2+.
  • Note: PKA’s activity peaks at 1 metal/ATP, declines beyond this.

5. General Base Catalysis

  • Asp166 proposed as general base but likely serves structural positioning.
  • Protein kinases do not require general-base catalysis, suggesting dissociative-like phosphoryl transfer without proton abstraction.

6. QM/MM Insights

  • Quantum mechanics/molecular mechanics (QM/MM) modeling needed to dissect reaction mechanisms—potential direction for RD’s study.

7. Rate-Determining Step

  • ADP release (product release) is the rate-limiting step for PKA:
    • Supported by Mg2+ dependence and viscosity studies.
  • Phosphoryl transfer is rapid.
  • Intrinsic ATPase activity suggests chemical step is not limiting.
  • Rate-determining step can differ by kinase and conditions (phosphoryl transfer, product release, or both).

Take-home Messages

  • Phosphoryl transfer by kinases is fast, dissociative, metal-dependent, and requires precise substrate positioning.
  • Activation loops regulate access and activity variably—sometimes like a “door,” but not universally.
  • ADP/product release is often rate-limiting, critical for understanding kinase turnover.
  • Metal ion dynamics are complex and kinase-specific—not always just “Mg2+ + ATP.”
  • Structural insights and QM/MM offer promising avenues to unravel unresolved catalytic mysteries.
  • Distal docking and substrate recognition extend beyond consensus sites—highlighting the holistic nature of kinase-substrate interactions.

RD’s Thoughts and Learnings

  • RD finds this paper deeply inspiring—dense but elegant in laying out the catalytic logic of kinases.
  • The role of Asp166 as a “non-classical” catalyst makes RD think more about transition states than simple acid/base chemistry.
  • Metal ion interplay and activation loop behavior remind RD of allosteric enzyme models—worth exploring more via QM/MM and kinetic modeling.
  • RD loves the idea of connecting distant structural changes to catalytic output—perfect for future studies on kinase regulation and phosphorylation dynamics.

To-Do/Future Ideas

  • Explore QM/MM modeling of PKA catalysis.
  • Deep dive into activation loop disorder-order transitions across kinase families.
  • Study metal ion-dependent activity modulation in diverse kinases (TPKs, CSK).
  • Examine rate-determining steps using rapid quench flow data and kinetic modeling.

I LOVE THIS PAPER! 💙