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 Ca(2+)-binding site to the N-terminal region of the enzyme. Thus, in a (45)Ca(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 Ca(2+)-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 Ca(2+) ligands. Indeed, mutations particularly in loop 2 (N43A, D44A, and E46A) led to decreased Ca(2+) binding and a requirement for higher Ca(2+) concentrations to stimulate enzyme activity. Our data indicate that an N-terminal beta-sandwich of 5-LO functions as a C2 domain in the calcium regulation of enzyme activity.
Hammarberg
T
T
Department of Medical Biochemistry and Biophysics, Divisions of Chemistry II and Chemistry I, and the Stockholm Bioinformatics Centre, Karolinska Institutet, SE-17177 Stockholm, Sweden.
Provost
P
P
Persson
B
B
Rådmark
O
O
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000737:chemistry; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D011993:Recombinant Fusion Proteins; D001092:Arachidonate 12-Lipoxygenase; D001094:Arachidonate 5-Lipoxygenase; D005982:Glutathione Transferase; D010741:Phospholipases A; D002118:Calcium