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J. Biol. Chem., Vol. 280, Issue 46, 38756-38766, November 18, 2005

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On the Relationships of Substrate Orientation, Hydrogen Abstraction, and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant^*

Gianguido Coffa, Ann N. Imber, Brendan C. Maguire1, Gurunathan Laxmikanthan¶, Claus Schneider, Betty J. Gaffney2, and Alan R. Brash3

From the Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the Departments of Biological Science and ^¶Chemistry & Biochemistry (Institute of Molecular Biophysics), Florida State University, Tallahassee, Florida 32306

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-³H- and 11S-³H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S, 15S- and 8S, 15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R, 15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.

Received for publication, May 3, 2005 , and in revised form, September 12, 2005.

^* This work was supported by National Institutes of Health Grants GM53638 (to A. R. B.) and GM65268 (to B. J. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Anatomy, University of California, San Francisco, CA 94143.

² To whom correspondence may be addressed: Dept. of Biological Sciences, Florida State University, Tallahassee, FL 32306. Tel.: 850-644-8547; Fax: 850-644-0481; E-mail: gaffney{at}bio.fsu.edu.

³ To whom correspondence may be addressed: Dept. of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-343-4495; Fax: 615-322-4707; E-mail: alan.brash{at}vanderbilt.edu.

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