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Loop movement and catalysis in creatine kinase.

Pan-Fen Wang1, Allen J Flynn, Michael J McLeish

  • 1College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, USA.

IUBMB Life
|July 23, 2005
PubMed
Summary
This summary is machine-generated.

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Structural analysis of Torpedo californica creatine kinase reveals key loop movements and a His66-Asp326 salt bridge critical for catalysis. Mutagenesis confirms His66

Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzymology

Background:

  • The crystal structure of Torpedo californica creatine kinase was determined at 2.1 A resolution.
  • The dimeric structure presented two distinct monomer forms: one bound to MgADP and another to a transition-state analogue complex (MgADP, nitrate, creatine).

Purpose of the Study:

  • To investigate the structural and functional significance of loop movements and specific residues in the creatine kinase active site.
  • To elucidate the role of the conserved PGHP motif, particularly His66, in creatine kinase catalysis.

Main Methods:

  • X-ray crystallography to determine the 2.1 A crystal structure.
  • Alanine-scanning mutagenesis of the PGHP motif residues.
  • Enzyme kinetics analysis (implied by pH-rate profile discussion).

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Main Results:

  • A striking difference between the two structures is the movement of two loops (residues 60-70 and 323-333) into the active site in the transition state structure, occluding it from solvent.
  • This occlusion is stabilized by a salt bridge between His66 and Asp326.
  • Alanine-scanning mutagenesis identified His66 as the only critical residue within the PGHP motif for creatine kinase activity.
  • Neither His66 nor Asp326 directly interact with substrates but are crucial for precise substrate alignment for phosphoryl group transfer.

Conclusions:

  • The His66-Asp326 salt bridge and associated loop movements are vital for stabilizing the transition state and facilitating phosphoryl group transfer in creatine kinase.
  • His66 plays a significant catalytic role, distinct from other guanidino kinases, and is essential for precise substrate alignment.
  • His66 and Asp326 are not responsible for the observed pKs in the pH-rate profile of HMCK.