title: Dimerization regulates the enzymatic activity of Escherichia coli outer membrane phospholipase A.
abstract: The outer membrane phospholipase A (OMPLA) of Escherichia coli is present in a dormant state in the cell envelope. The enzyme is activated by various processes, which have in common that they perturb the outer membrane. Kinetic experiments, chemical cross-linking, and analytical ultracentrifugation were carried out with purified, detergent-solubilized OMPLA to understand the underlying mechanism that results in activation. Under conditions in which the enzyme displayed full activity, OMPLA was dimeric. High detergent concentrations or very dilute protein concentrations resulted in low specific activity of the enzyme, and under those conditions the enzyme was monomeric. The cofactor Ca2+ was required for dimerization. Covalent modification of the active site serine with hexadecylsulfonylfluoride resulted in stabilization of the dimeric form and a loss of the absolute calcium requirement for dimerization. The results of these experiments provide evidence for dimerization as the molecular mechanism by which the enzymatic activity of OMPLA is regulated. This dimerization probably plays a role in vivo as well. Data from chemical cross-linking on whole cells indicate that OMPLA is present in the outer membrane as a monomer and that activation of the enzyme induces dimerization concurrent with the appearance of enzymatic activity.
authors.affiliation: Department of Enzymology and Protein Engineering, Center for Biomembranes and Lipid Enzymology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. n.dekker@chem.ruu.nl
authors.forename: N
authors.initials: N
authors.lastname: Dekker
authors.forename: J
authors.initials: J
authors.lastname: Tommassen
authors.forename: A
authors.initials: A
authors.lastname: Lustig
authors.forename: J P
authors.initials: JP
authors.lastname: Rosenbusch
authors.forename: H M
authors.initials: HM
authors.lastname: Verheij
mesh_terms: D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005976:Glutaral; D008970:Molecular Weight; D010741:Phospholipases A / Q000378:metabolism*; D011487:Protein Conformation; D014461:Ultracentrifugation
chemical_list: D003902:Detergents; D010741:Phospholipases A; D005976:Glutaral