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Volume 271, Number 41, Issue of October 11, 1996 pp. 25412-25418
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Defining the Arachidonic Acid Binding Site of Human 15-Lipoxygenase
MOLECULAR MODELING AND MUTAGENESIS

(Received for publication, March 20, 1996, and in revised form, August 1, 1996)

Qing-Fen Gan , Michelle F. Browner , David L. Sloane and Elliott Sigal ^§

From Roche Bioscience, Palo Alto, California 94304,  Targeted Genetics, Seattle, Washington 98108, and ^§ Mercator Genetics, Menlo Park, California 94025

Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory disorders and are, therefore, important targets for drug discovery. Both plant and mammalian lipoxygenases catalyze the dioxygenation of polyunsaturated fatty acids, which contain one or more 1,4-cis,cis-pentadiene units to yield hydroperoxide products. At the time this study was initiated, soybean lipoxygenase-1 was the only lipoxygenase for which an atomic resolution structure had been determined. No structure of lipoxygenase with substrate or inhibitor bound is currently available. A model of arachidonic acid docked into the proposed substrate binding site in the soybean structure is presented here. Analysis of this model suggested two residues, an aromatic residue and a positively charged residue, could be critical for substrate binding. Validation of this model is provided by site-directed mutagenesis of human 15-lipoxygenase, despite the low amino acid sequence identity between the soybean and mammalian enzymes. Both a positively charged amino acid residue (Arg⁴⁰²) and an aromatic amino acid residue (Phe⁴¹⁴) are identified as critical for the binding of fatty acid substrates in human 15-lipoxygenase. Thus, binding determinants shown to be characteristic of non-enzymatic fatty acid-binding proteins are now implicated in the substrate binding pocket of lipoxygenases.

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