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Why is creatine kinase a dimer? Evidence for cooperativity between the two subunits
T Hornemann1, D Rutishauser, T Wallimann
1Swiss Federal Institute of Technology, Institute of Cell Biology HPM F44, ETHZ-Hönggerberg, 8093, Zürich, Switzerland. horneman@cell.biol.ethz.ch
Biochimica Et Biophysica Acta
|July 19, 2000
Summary
Mutating chicken brain creatine kinase (BB-CK) reduced its activity. Hybridizing with muscle enzyme (MM-CK) revealed subunit cooperativity in ATP synthesis but independent function in phosphocreatine synthesis.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Creatine kinase (CK) exists as different isoenzymes, including brain (BB-CK) and muscle (MM-CK).
- The catalytic site of BB-CK is crucial for its enzymatic activity.
- Understanding isoenzyme function and subunit interactions is key to comprehending cellular energy buffering.
Purpose of the Study:
- To investigate the role of individual subunits in creatine kinase (CK) activity.
- To explore subunit cooperativity in different reaction directions using engineered CK hybrids.
- To characterize the kinetic parameters of wild-type and mutated CK isoenzymes and their hybrids.
Main Methods:
- Site-directed mutagenesis (C283S) was used to inactivate chicken BB-CK.
- Hybridization of mutated BB-CK with native MM-CK and his-tagged BB-CK to form heterodimeric and homodimeric hybrids.
- Purification of enzyme hybrids using chromatography.
- Enzymatic assays to determine kinetic parameters (Vmax, Km, Kd) for both forward (phosphocreatine synthesis) and reverse (ATP synthesis) reactions.
Main Results:
- The C283S mutation in BB-CK resulted in a catalytically inactive dimer (B*B*-CK) with residual activity of ~4%.
- MB*-CK and hBB*-CK hybrids showed significantly reduced Vmax in the ATP synthesis direction (reverse reaction) compared to wild-type.
- Inactivation of one subunit in MB*-CK led to increased Kd and substrate synergism in the reverse reaction, indicating cooperativity.
- In the phosphocreatine synthesis direction (forward reaction), hybrids exhibited reduced Vmax but unaltered Km and Kd, suggesting independent subunit function.
Conclusions:
- Enzymatic cooperativity between subunits is evident in the reverse reaction (ATP synthesis) of creatine kinase.
- Subunits function independently in the forward reaction (phosphocreatine synthesis).
- Mutational analysis and hybridization provide insights into the complex regulatory mechanisms of creatine kinase isoenzymes.