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Activation of a covalent outer membrane phospholipase A dimer
Roelie L Kingma1, Maarten R Egmond
1Department of Membrane Enzymology, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, the Netherlands.
European Journal of Biochemistry
|May 3, 2002
Summary
Outer membrane phospholipase A (OMPLA) activity requires a proper dimer interface for function. Covalent OMPLA dimers highlight the essential role of dimer formation in regulating enzyme activity and calcium binding.
Area of Science:
- Biochemistry
- Enzymology
- Membrane Biology
Background:
- Outer membrane phospholipase A (OMPLA) activity is regulated by reversible dimerization.
- Native OMPLA in phospholipid vesicles exists as a dimer but is inactive.
Purpose of the Study:
- Investigate the importance of dimerization for controlling OMPLA activity.
- Compare properties of a covalent OMPLA dimer to native OMPLA.
Main Methods:
- Constructed a covalent OMPLA dimer.
- Compared native and covalent OMPLA in micellar detergent and phospholipid bilayers.
- Assessed OMPLA activity and calcium binding affinity.
Main Results:
- Covalent OMPLA dimer activity was detergent-independent in micelles.
- Covalent OMPLA dimer showed high calcium affinity in micelles but required high calcium in vesicles.
- Vesicle solubilization increased calcium affinity, indicating improper dimer interface in bilayers.
- Impaired dimer interface variants lacked high affinity calcium binding.
Conclusions:
- A proper dimer interface is essential for optimal OMPLA catalysis.
- Covalent linkage could not restore function to variants with impaired dimer interfaces.
- Dimer interface formation is critical for high-affinity calcium binding and enzyme activity.