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

Human 5-lipoxygenase (5-LO) is a key enzyme in the conversion of arachidoni c acid into leukotrienes and lipoxins, mediators and modulators of inflamma tion. 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-te rminal 128 amino acids of recombinant human 5-LO (fused to glutathione S-tr ansferase) bound radioactive calcium to about the same extent as intact 5-L O. 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 ret iculocyte 15-LO. This model resembles beta -sandwich C2 domains of other Ca 2+-binding proteins. Comparison of our model with the C2 domain of cytosoli c phospholipase A(2) suggested a number of amino acids, located in the loop s that connect the beta -strands, as potential Ca2+ ligands. Indeed, mutati ons particularly in loop 2 (N43A, D44A, and E46A) led to decreased Ca2+ bin ding and a requirement for higher Ca2+ concentrations to stimulate enzyme a ctivity. Our data indicate that an N-termind beta -sandwich of 5-LO functio ns as a C2 domain in the calcium regulation of enzyme activity.