The N-terminal domain of 5-lipoxygenase binds calcium and mediates calcium stimulation of enzyme activity

Human 5-lipoxygenase (5-LO) is a key enzyme in the conversion of arachidonic acid into leukotrienes and lipoxins, mediators and modulators of inflammation. In this study, we localized a stimulatory Ca2+-binding site to the N-terminal region of the enzyme. Thus, in a Ca-45(2+) overlay assay, the N-terminal 128 amino acids of recombinant human 5-LO (fused to glutathione S-transferase) bound radioactive calcium to about the same extent as intact 5-LO. The glutathione S-transferase fusion protein of the C-terminal part of 5-LO (amino acids 120-673) showed much weaker binding. A model of a putative 5-LO N-terminal domain was calculated based on the structure of rabbit reticulocyte 15-LO. This model resembles beta -sandwich C2 domains of other Ca2+-binding proteins. Comparison of our model with the C2 domain of cytosolic phospholipase A(2) suggested a number of amino acids, located in the loops that connect the beta -strands, as potential Ca2+ ligands. Indeed, mutations particularly in loop 2 (N43A, D44A, and E46A) led to decreased Ca2+ binding and a requirement for higher Ca2+ concentrations to stimulate enzyme activity. Our data indicate that an N-termind beta -sandwich of 5-LO functions as a C2 domain in the calcium regulation of enzyme activity.