Activation of a covalent outer membrane phospholipase A dimer.
The activity of outer membrane phospholipase A (OMPLA) is regulated by reversible dimerization. However, native OMPLA reconstituted in phospholipid vesicles was found to be present as a dimer but nevertheless inactive. To investigate the importance of dimerization for control of OMPLA activity, a covalent OMPLA dimer was constructed and its properties were compared to native OMPLA both in a micellar detergent and after reconstitution in a phospholipid bilayer. Unlike native OMPLA, activity of the covalent OMPLA dimer was independent of type and concentration of detergent in micellar systems. In such systems, the covalent OMPLA dimer invariantly displayed high calcium affinity. In contrast, high calcium concentrations were required to activate a covalent OMPLA dimer when present in intact vesicles. Solubilization of the vesicles increased the affinity for calcium, suggesting that in an intact bilayer the dimer interface is not properly formed. This was supported by the observation that OMPLA variants having an impaired dimeric interface also lacked high affinity calcium binding. A covalent linkage was not able to restore high affinity calcium binding in these variants, demonstrating that a proper dimer interface is essential for optimal catalysis.
Kingma
Roelie L
RL
Department of Membrane Enzymology, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, the Netherlands.
Egmond
Maarten R
MR
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D003432:Cross-Linking Reagents; D003902:Detergents; D019281:Dimerization; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054466:Phospholipases A1
D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D003902:Detergents; D010741:Phospholipases A; D054466:Phospholipases A1; C412876:outer membrane phospholipase A; D002118:Calcium