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Nucleotide binding to creatine kinase: an isothermal titration microcalorimetry study
M Forstner1, C Berger, T Wallimann
1Institute of Cell Biology, Swiss Federal Institute of Technology Zürich, ETH Hönggerberg, CH-8093, Zürich, Switzerland. michael@xray.bmc.uu.se
FEBS Letters
|November 24, 1999
Summary
Muscle creatine kinase (CK) binding of ATP, with or without Mg(2+), involves no proton exchange and occurs only at active sites. The study found no interdependence between the dimeric CK active sites.
Area of Science:
- Biochemistry
- Enzymology
- Protein-ligand interactions
Background:
- Muscle creatine kinase (CK) is crucial for cellular energy homeostasis.
- Understanding ATP binding is key to elucidating CK's enzymatic mechanism.
- Dimeric structure of CK suggests potential for inter-site communication.
Purpose of the Study:
- To thermodynamically characterize ATP binding to dimeric muscle creatine kinase (CK).
- To investigate the role of Mg(2+) in ATP binding to CK.
- To determine the influence of pH and temperature on CK-ATP interactions.
Main Methods:
- Isothermal titration microcalorimetry (ITC) was employed to measure binding thermodynamics.
- Experiments were conducted across varying pH and temperature conditions.
- A thermodynamic cycle was utilized to analyze domain movement contributions.
Main Results:
- ATP binding to CK active sites does not involve proton exchange with the buffer.
- Nucleotide binding is exclusively localized to the active sites of CK.
- No evidence of interdependence between the active sites of the dimeric CK was observed.
- Major structural changes occur upon Mg-ATP binding to CK.
Conclusions:
- The thermodynamic data clarify the nature of nucleotide binding to muscle creatine kinase.
- Mg(2+) influences the structural dynamics of CK upon nucleotide binding.
- The study provides insights into the functional and structural properties of dimeric CK.