Creatine KinaseCreatine Kinase

Amy WardAmy Ward

OverviewOverview

Metabolism Metabolism Creatine Kinase IsoformsCreatine Kinase Isoforms ATP RecyclingATP Recycling Clinical RelevanceClinical Relevance

MetabolismMetabolism

ATP is the energy currency in the cellATP is the energy currency in the cell Cellular respiration occurs in the Cellular respiration occurs in the

mitochondriamitochondria Muscle and brain are most actively Muscle and brain are most actively

metabolizing tissuesmetabolizing tissues

ATP as Energy SourceATP as Energy Source

ATP donates high energy bond in ATP donates high energy bond in coupled reactionscoupled reactions

Substrate ProductSubstrate Product

ATP ADPATP ADP

ATP RecyclingATP Recycling

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

Energy homeostasisEnergy homeostasis

PCr Cr PCr Cr

ADPADP ATPATP

Creatine KinaseCreatine Kinase

Crystallization attempts date back to Crystallization attempts date back to 1950s1950s

First successful crystal formed in 1996First successful crystal formed in 1996

Creatine KinaseCreatine Kinase

Different isoforms depending on locationDifferent isoforms depending on location Coupled to sites of energy production or Coupled to sites of energy production or

consumptionconsumption

CK IsoformsCK Isoforms

Cytosolic IsoformsCytosolic Isoforms Muscle-typeMuscle-type Brain-typeBrain-type

Exist as dimersExist as dimers Temporal energy bufferingTemporal energy buffering

Mitochondrial IsoformsMitochondrial Isoforms Exist in dimer-octamer equilibriumExist in dimer-octamer equilibrium Spatial energy bufferingSpatial energy buffering

Cytosolic IsoformsCytosolic Isoforms

Subunits: M and BSubunits: M and B Dimeric isoenzymes in cytosol (85 kDa):Dimeric isoenzymes in cytosol (85 kDa):

MM (muscle-type)MM (muscle-type) BB (brain-type)BB (brain-type) MB hybridMB hybrid

Cytosolic IsoformsCytosolic Isoforms

Function as a temporal energy bufferFunction as a temporal energy buffer ADP + PCr ADP + PCr ATP + Cr ATP + Cr Coupled to:Coupled to:

GlycolysisGlycolysis Actin-myosin systemActin-myosin system

Temporal Energy BufferingTemporal Energy Buffering

Muscle-Type CK: Muscle-Type CK: MonomerMonomer

Small N Small N domaindomain

Large C Large C domaindomain

Muscle-Type CKMuscle-Type CK

Muscle-Type CK: DimerMuscle-Type CK: Dimer

Monomer-monomer Monomer-monomer interface site highly interface site highly conservedconserved All isoenzymes have:All isoenzymes have:

4 Trp sites4 Trp sites 4 Cys sites4 Cys sites

Muscle-Type CKMuscle-Type CK

MM-CK bound to M-MM-CK bound to M-band in myofibrilband in myofibril

Cardiac tissue: 50% Cardiac tissue: 50% of CK actionof CK action

Muscle-type CKMuscle-type CK

CK maintains high CK maintains high ATP concentrationATP concentration

Muscle-Type CKMuscle-Type CK

Mutation in CK genes linked to Mutation in CK genes linked to myocardial infarctionmyocardial infarction

Heart diseases linked to low levels of CK Heart diseases linked to low levels of CK

Brain-Type CKBrain-Type CK

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

BB BB MB MB MM MM

Brain-Type CKBrain-Type CK

CK levels associated with learning CK levels associated with learning processesprocesses

CK overexpressed in tumoursCK overexpressed in tumours Decreased CK Decreased CK neurodegeneration neurodegeneration

Mitochondrial CKMitochondrial CK

Bound to outside of inner membrane Bound to outside of inner membrane

within cristaewithin cristae Form microcompartments with porinsForm microcompartments with porins

Mitochondrial CKMitochondrial CK

TransphosphorylationTransphosphorylation Cr enters through poreCr enters through pore Cr + ATP Cr + ATP PCr + ADP PCr + ADP PCr exits through porePCr exits through pore

PCr mediates between sites of ATP PCr mediates between sites of ATP consumption and productionconsumption and production

Spatial Energy BufferingSpatial Energy Buffering

Mitochondrial CKMitochondrial CK

Mi-CK: StructureMi-CK: Structure

Mi-CK: MonomerMi-CK: Monomer

Small (residues 1-112) N-terminal domainSmall (residues 1-112) N-terminal domain Large (residues 113-380) C-terminal domainLarge (residues 113-380) C-terminal domain ATP binding site located in the cleft between ATP binding site located in the cleft between

the two domainsthe two domains

Mi-CK: Dimer Mi-CK: Dimer

Trp residuesTrp residues Trp 206: monomer-Trp 206: monomer-

monomer contactmonomer contact Trp 264 & N-Trp 264 & N-

terminal: octamer terminal: octamer formingforming

Mi-CK: OctamerMi-CK: Octamer

stable against denaturation stable against denaturation insensitive to proteolysisinsensitive to proteolysis Dissociation to dimer takes hours to Dissociation to dimer takes hours to

weeksweeks Accelerated with addition of transition Accelerated with addition of transition

state analogue, TSAC = creatine, Mg-state analogue, TSAC = creatine, Mg-ADP & nitrateADP & nitrate

Mi-CK: StructureMi-CK: Structure

Mi-CK fold differs from all other kinasesMi-CK fold differs from all other kinases Structures of Mi-CK-ATP and free Structures of Mi-CK-ATP and free

enzyme very similarenzyme very similar

Mi-CK: StructureMi-CK: Structure

Active site residues:Active site residues: Phosphate groups of ATP interact with Arg Phosphate groups of ATP interact with Arg

residues 125, 127, 287, 315residues 125, 127, 287, 315 Cys278: substrate bindingCys278: substrate binding His61: mutation impairs enzyme activityHis61: mutation impairs enzyme activity Loop residues 60-65 moves toward active Loop residues 60-65 moves toward active

site for catalysissite for catalysis Trp223: crucial for catalysisTrp223: crucial for catalysis

Mi-CK: Octameric Mi-CK: Octameric StructureStructure

Mi-CK: Octameric Mi-CK: Octameric StructureStructure

ATP RecyclingATP Recycling

The PCr circuit:The PCr circuit: Spatial separation of Spatial separation of

ATP consumption ATP consumption and synthesisand synthesis

Mitochondrial VS Mitochondrial VS Cytosolic CKCytosolic CK

Very similar structures and structural Very similar structures and structural elementselements

Mi-CK evolved different folding pattern Mi-CK evolved different folding pattern for catalyzing phosphoryl transferfor catalyzing phosphoryl transfer

Allow compartmentalization of functionAllow compartmentalization of function

ReferencesReferences

1. Wallimann T et al. 1998. Some new aspects of creatine kinase (CK): compartmentation, 1. Wallimann T et al. 1998. Some new aspects of creatine kinase (CK): compartmentation, structure, function and regulation for cellular and mitochondrial bioenergetics and structure, function and regulation for cellular and mitochondrial bioenergetics and physiology. physiology. BiofactorsBiofactors 88, 229-234., 229-234.

2. Schlattner U et al. 1998. Functional aspects of the X-ray structure of mitochondrial creatine 2. Schlattner U et al. 1998. Functional aspects of the X-ray structure of mitochondrial creatine kinase: A molecular physiology approach. kinase: A molecular physiology approach. Molecular and Cellular Biochemistry Molecular and Cellular Biochemistry 184184, 125-, 125-140. 140.

3. Yamamichi H et al. 2001. Creatine kinase gene mutation in a patient with muscle creatine 3. Yamamichi H et al. 2001. Creatine kinase gene mutation in a patient with muscle creatine kinase deficiency. kinase deficiency. Clinical Chemistry Clinical Chemistry 4747, 1967-1973., 1967-1973.

4. Alberts B et al. 1994. Molecular Biology of the Cell, 34. Alberts B et al. 1994. Molecular Biology of the Cell, 3 rdrd edition. New York: Garland Publishing. edition. New York: Garland Publishing.

5. Lipskaya TY. 2000. The physiological role of the creatine kinase system: evolution of views. 5. Lipskaya TY. 2000. The physiological role of the creatine kinase system: evolution of views. Biochemistry (Moscow) Biochemistry (Moscow) 6666, 115-129. , 115-129.