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Domain closure in adenylate kinase
M A Sinev1, E V Sineva, V Ittah
1Department of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.
Biochemistry
|May 21, 1996
Summary
This study used time-resolved excitation energy transfer to show adenylate kinase undergoes a two-step domain closure upon ligand binding. This reveals multiple enzyme conformations in solution.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Adenylate kinase (AK) is crucial for cellular energy homeostasis.
- Understanding AK's conformational changes is key to its function.
- Previous studies proposed domain closure in AK based on crystal structures.
Purpose of the Study:
- To investigate the proposed domain closure mechanism of adenylate kinase (AK).
- To quantitatively analyze the enzyme's conformational changes in solution.
- To confirm the stepwise nature of domain closure using a biophysical technique.
Main Methods:
- Time-resolved nonradiative excitation energy transfer (ET) was employed.
- A site-specifically labeled mutant of Escherichia coli AK was engineered.
- The mutant enzyme was studied in various ligand-bound states (apo, MgATP, AMP, AP5A).
Main Results:
- Excitation energy transfer efficiencies increased with ligand binding, indicating decreasing distances.
- Intermolecular distance distributions were calculated, showing significant reduction.
- Mean distances decreased from 31 Å (apo) to 12 Å (AP5A complex).
Conclusions:
- The results confirm a stepwise, two-step domain closure mechanism for AK.
- Multiple conformations of E. coli AK exist in solution.
- The study provides dynamic insights into enzyme conformational flexibility.