1. Creatine Kinase Amy Ward

2. Overview  Metabolism  Creatine Kinase Isoforms  ATP Recycling  Clinical Relevance

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

4. ATP as Energy Source  ATP donates high energy bond in coupled reactions  Substrate Product ATP ADP ATP ADP

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

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

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

9. CK Isoforms  Cytosolic Isoforms  Muscle-type  Brain-type  Exist as dimers  Temporal energy buffering  Mitochondrial Isoforms  Exist in dimer-octamer equilibrium  Spatial energy buffering

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

11. Cytosolic Isoforms  Function as a temporal energy buffer  ADP + PCr  ATP + Cr  Coupled to:  Glycolysis  Actin-myosin system  Temporal Energy Buffering

12. Muscle-Type CK: Monomer  Small N domain  Large C domain

13. Muscle-Type CK

14. Muscle-Type CK: Dimer  Monomer-monomer interface site highly conserved  All isoenzymes have:  4 Trp sites  4 Cys sites

15. Muscle-Type CK  MM-CK bound to M- band in myofibril  Cardiac tissue: 50% of CK action

16. Muscle-type CK  CK maintains high ATP concentration

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

18. 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

19. Brain-Type CK  CK levels associated with learning processes  CK overexpressed in tumours  Decreased CK  neurodegeneration

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

21. 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

22. Mitochondrial CK

23. Mi-CK: Structure

24. 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

25. Mi-CK: Dimer  Trp residues  Trp 206: monomer- monomer contact  Trp 264 & N- terminal: octamer forming

26. 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, Mg- ADP & nitrate

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

28. 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

29. Mi-CK: Octameric Structure

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

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

33. 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, 125- 140. 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, 3 rd 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.