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Tryptophan 500 and Arginine 707 Define Product and Substrate Active Site Binding in Soybean Lipoxygenase-1^†

Viola C. Ruddat,^‡ Rakesh Mogul,^‡ Ilya Chorny,^# Cameron Chen,^‡ Noah Perrin,^‡ Stephanie Whitman,^‡ Victor Kenyon,^‡ Matthew P. Jacobson,^# Claude F. Bernasconi,^‡ and Theodore R. Holman*^‡

Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, and Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-2240

Biochemistry, 2004, 43 (41), pp 13063–13071

DOI: 10.1021/bi0489098

Publication Date (Web): September 25, 2004

†

This research has been supported NIH Grant GM56062-06 (TRH). I.C. is supported by a postdoctoral fellowship from the Alfred P. Sloan foundation and the U.S. Department of Energy.

‡

Department of Chemistry and Biochemistry, University of California, Santa Cruz.

Department of Pharmaceutical Chemistry, University of California, San Francisco.

To whom correspondence should be sent. Phone: (831) 459-5884; fax: (831) 459-2935; e-mail: tholman@chemistry.ucsc.edu.

Abstract

There is much debate whether the fatty acid substrate of lipoxygenase binds “carboxylate-end first” or “methyl-end first” in the active site of soybean lipoxygenase-1 (sLO-1). To address this issue, we investigated the sLO-1 mutants Trp500Leu, Trp500Phe, Lys260Leu, and Arg707Leu with steady-state and stopped-flow kinetics. Our data indicate that the substrates (linoleic acid (LA), arachidonic acid (AA)), and the products (13-(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (HPOD) and 15-(S)-hydroperoxyeicosatetraeonic acid (15-(S)-HPETE)) interact with the aromatic residue Trp500 (possibly π−π interaction) and with the positively charged amino acid residue Arg707 (charge−charge interaction). Residue Lys260 of soybean lipoxygenase-1 had little effect on either the activation or steady-state kinetics, indicating that both the substrates and products bind “carboxylate-end first” with sLO-1 and not “methyl-end first” as has been proposed for human 15-lipoxygenase.

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