Keywords

Kinase, Phosphorylation, Catalysis, Mechanism, SN1, SN2, Binding

Reference

DOI: 10.1016/s0969-2126(98)00043-4

Abstract

Protein kinases are essential enzymes involved in phosphoryl transfer, pivotal to many signaling processes.
Crystal structures of kinase-substrate complexes reveal critical structural features for catalysis.
Recent structural studies mimicking transition state complexes provide insights into whether kinases use associative (SN2-like) or dissociative (SN1-like) phosphoryl transfer mechanisms.

Notes

1. Conformational Changes in Kinases

  • Global changes:
    • Many kinases exhibit hinge-bending or domain rotation for active site closure and precise substrate positioning.
    • E.g., adenylate kinase, phosphoglycerate kinase.
  • Local shifts:
    • Small adjustments like shear motion in E. coli phosphofructokinase.
    • Glycine-rich loop in PKA flexibly interacts with ATP.
  • Minimal changes:
    • Some kinases (e.g., nucleoside diphosphate kinase) show little conformational adjustment upon substrate binding, reflecting diverse mechanistic strategies.
  • Binding may induce changes or select pre-existing conformations, supporting conformational selection models.

2. Cofactor Roles: Mg²⁺ and Mn²⁺

  • Mg²⁺–nucleotide complex is essential for catalysis, coordinating β- and γ-phosphoryl groups and aligning substrates for transfer.
  • Water molecules and protein oxygens complete Mg²⁺ coordination sphere, promoting a favorable geometry for phosphoryl transfer.
  • In ADP/GDP-bound kinases, Mg²⁺ bridges β-phosphate and phosphorylated product, aiding catalysis.
  • Some kinases require two divalent cations:
    • E.g., pyruvate kinase, phosphoenolpyruvate carboxykinase—Mg²⁺ for nucleotide binding, Mn²⁺ for catalysis.
  • Phosphoenolpyruvate carboxykinase crystal structure shows Mn²⁺ bridging ATP and pyruvate, coordinating transition states and intermediates.

3. Binding Mechanisms: Motifs and Specific Interactions

  • Nucleotide binding via P-loop (phosphate-binding loop):
    • Conserved glycines allow close approach to phosphoryl groups.
    • Ser/Thr residues coordinate Mg²⁺.
    • Lysine stabilizes β- and γ-phosphates.
  • Adenine binding:
    • Less conserved, often via hydrophobic interactions or main-chain hydrogen bonds.
    • Structural water may assist nucleotide stabilization.
  • α-helix dipole may stabilize phosphoryl groups, enhancing transfer readiness.

4. Catalytic Mechanisms: Single vs. Double Displacement

  • Single displacement (direct displacement):
    • Ternary complex forms with both substrates bound before transfer.
  • Double displacement (ping-pong):
    • Formation of phospho-enzyme intermediate (e.g., via histidine) before transferring phosphate to acceptor.

5. Phosphoryl Transfer Mechanisms: SN1 vs. SN2

  • Associative (SN2-like):
    • Direct nucleophilic attack, forming a pentacoordinate phosphorane (PO₄³⁻).
    • Transition state with net charge ~−3.
    • Short distances between phosphate and acceptor, promoting in-line attack.
  • Dissociative (SN1-like):
    • Phosphate leaves first, forming planar metaphosphate (PO₃⁻), net charge ~−1.
    • Nucleophile attacks after phosphate group dissociates, stepwise bond formation.
  • Conflicting evidence:
    • UMP/CMP kinase supports associative mechanism (SN2-like).
    • Nucleoside diphosphate kinase, H-Ras show mixed features, unclear dominant mechanism.

6. RD’s Thoughts and Takeaways

  • The paper beautifully connects structural data to chemical mechanism, clarifying how kinases achieve efficient phosphate transfer.
  • Conformational flexibility, cofactor orchestration, and precise binding motifs are all interdependent features enabling catalysis.
  • The SN1 vs. SN2 debate in kinases is nuanced, and context- or kinase-dependent—important for RD’s own understanding of catalysis.
  • P-loop’s conserved glycines show how minimal side-chain bulk is critical to allow phosphoryl groups to be held close for reaction.
  • RD appreciates the depth on metal ion roles (Mg²⁺, Mn²⁺)—not just structural but dynamic in catalysis.
  • The analogy to adenylate and phosphoglycerate kinase helps link domain motion with catalytic readiness.

Take-home Messages

  • Kinase conformational changes are vital for precise phosphoryl transfer.
  • Divalent cations (Mg²⁺, Mn²⁺) play active roles in substrate alignment and transition state stabilization.
  • Phosphate binding relies on P-loop and lysine-mediated motifs, while adenine binding is more variable.
  • Both SN1 and SN2-like features are present in kinase-catalyzed phosphate transfer; mechanism may vary between kinases.
  • The balance of structural flexibility and precise interactions allows kinases to efficiently manage phosphate transfer reactions critical for signaling.