Creatine Kinase

Amy Ward

Overview
Metabolism Creatine Kinase Isoforms ATP Recycling Clinical Relevance

Metabolism
ATP is the energy currency in the cell Cellular respiration occurs in the mitochondria Muscle and brain are most actively metabolizing tissues

ATP as Energy Source

ATP donates high energy bond in coupled reactions
Substrate
ATP

Product
ADP

ATP Recycling
Creatine kinase catalyzes transfer of phosphate from N-phosphoryl creatine (PCr) to ADP Energy homeostasis
PCr
ADP

Cr
ATP

Creatine Kinase
Crystallization attempts date back to 1950s First successful crystal formed in 1996

Creatine Kinase
Different isoforms depending on location Coupled to sites of energy production or consumption

CK Isoforms
Cytosolic Isoforms
Muscle-type Brain-type

Exist as dimers Temporal energy buffering

Mitochondrial Isoforms
Exist in dimer-octamer equilibrium Spatial energy buffering

Cytosolic Isoforms
Subunits: M and B Dimeric isoenzymes in cytosol (85 kDa):
MM (muscle-type) BB (brain-type) MB hybrid

Cytosolic Isoforms
Function as a temporal energy buffer ADP + PCr ATP + Cr Coupled to:
Glycolysis Actin-myosin system

Temporal Energy Buffering

Muscle-Type CK: Monomer

Small N domain Large C domain

Muscle-Type CK

Muscle-Type CK: Dimer

Monomer-monomer interface site highly conserved
All isoenzymes have:
4 Trp sites 4 Cys sites

Muscle-Type CK
MM-CK bound to Mband in myofibril Cardiac tissue: 50% of CK action

Muscle-type CK
CK maintains high ATP concentration

Muscle-Type CK
Mutation in CK genes linked to myocardial infarction Heart diseases linked to low levels of CK

Brain-Type CK
Structure very similar to Muscle-Type CK Most tissues contain MB and BB types High levels in brain, retina, and sperm BB form is the precursor for the other two
BB MB MM

Brain-Type CK
CK levels associated with learning processes CK overexpressed in tumours Decreased CK neurodegeneration

Mitochondrial CK
Bound to outside of inner membrane within cristae Form microcompartments with porins

Mitochondrial CK
Transphosphorylation
Cr enters through pore Cr + ATP PCr + ADP PCr exits through pore

PCr mediates between sites of ATP consumption and production Spatial Energy Buffering

Mitochondrial CK

Mi-CK: Structure

Mi-CK: Monomer
Small (residues 1-112) N-terminal domain Large (residues 113-380) C-terminal domain ATP binding site located in the cleft between the two domains

Mi-CK: Dimer
Trp residues
Trp 206: monomermonomer contact Trp 264 & Nterminal: octamer forming

Mi-CK: Octamer
stable against denaturation insensitive to proteolysis Dissociation to dimer takes hours to weeks Accelerated with addition of transition state analogue, TSAC = creatine, MgADP & nitrate

Mi-CK: Structure
Mi-CK fold differs from all other kinases Structures of Mi-CK-ATP and free enzyme very similar

Mi-CK: Structure
Active site residues:
Phosphate groups of ATP interact with Arg residues 125, 127, 287, 315 Cys278: substrate binding His61: mutation impairs enzyme activity Loop residues 60-65 moves toward active site for catalysis Trp223: crucial for catalysis

Mi-CK: Octameric Structure

Mi-CK: Octameric Structure

ATP Recycling
The PCr circuit:
Spatial separation of ATP consumption and synthesis

Mitochondrial VS Cytosolic CK
Very similar structures and structural elements Mi-CK evolved different folding pattern for catalyzing phosphoryl transfer Allow compartmentalization of function

References
1. Wallimann T et al. 1998. Some new aspects of creatine kinase (CK): compartmentation, structure, function and regulation for cellular and mitochondrial bioenergetics and physiology. Biofactors 8, 229-234. 2. Schlattner U et al. 1998. Functional aspects of the X-ray structure of mitochondrial creatine kinase: A molecular physiology approach. Molecular and Cellular Biochemistry 184, 125140. 3. Yamamichi H et al. 2001. Creatine kinase gene mutation in a patient with muscle creatine kinase deficiency. Clinical Chemistry 47, 1967-1973. 4. Alberts B et al. 1994. Molecular Biology of the Cell, 3rd edition. New York: Garland Publishing.

5. Lipskaya TY. 2000. The physiological role of the creatine kinase system: evolution of views. Biochemistry (Moscow) 66, 115-129.