Investigations into calcium-dependent membrane association of 15-lipoxygenase-1 - Mechanistic roles of surface-exposed hydrophobic amino acids and calcium

Among mammalian lipoxygenases the 15-lipoxygenase-1 is somewhat special because of its capability of oxygenating complex lipid-protein assemblies (biomembranes, lipoproteins) and previous investigations have implicated calcium in enzyme/membrane interaction. We investigated the mechanism of calcium-dependent membrane association and obtained the following results. (i) Membrane binding of 15-lipoxygenase-1 involves electrostatic forces as well as hydrophobic interactions of solvent-exposed apolar amino acids (Tyr(15), Phe(70), Leu(71), Trp(181), and Leu(195)) with the hydrophobic core of membrane phospholipids. These sequence determinants of membrane association are clustered at the membrane contact plane of the enzyme that also involves the entrance to the substrate binding pocket. Site-directed mutagenesis of these determinants to negatively charged residues strongly impaired membrane binding. (ii) Calcium at micromolar concentrations (5-50 muM) is required for efficient membrane binding. For direct 15-lipoxygenase/calcium interaction a dissociation constant of 2-5x10(-4) M was determined (low affinity binding) and we failed to detect high affinity calcium-binding sites at the enzyme. Reversible low affinity calcium binding induces subtle structural alterations of the enzyme, which did not impact catalytic activity. (iii) Increasing calcium concentrations failed to reverse impairment of membrane binding induced by mutagenesis of the sequence determinants indicating the priority of hydrophobic interactions. Taken together these data suggest that 15-lipoxygenase-1 associates to biomembranes primarily via hydrophobic interactions between surface-exposed apolar amino acid side chains and membrane lipids. Calcium supports membrane binding probably by forming salt bridges between the negatively charged head groups of membrane phospholipids and acidic surface amino acids of the membrane contact plane and this interaction might contribute to overcome repulsive forces.