title: On the relationships of substrate orientation, hydrogen abstraction, and product stereochemistry in single and double dioxygenations by soybean lipoxygenase-1 and its Ala542Gly mutant.
abstract: 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-(3)H- and 11S-(3)H-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.
authors.affiliation: Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Gianguido
authors.initials: G
authors.lastname: Coffa
authors.forename: Ann N
authors.initials: AN
authors.lastname: Imber
authors.forename: Brendan C
authors.initials: BC
authors.lastname: Maguire
authors.forename: Gurunathan
authors.initials: G
authors.lastname: Laxmikanthan
authors.forename: Claus
authors.initials: C
authors.lastname: Schneider
authors.forename: Betty J
authors.initials: BJ
authors.lastname: Gaffney
authors.forename: Alan R
authors.initials: AR
authors.lastname: Brash
mesh_terms: D000409:Alanine / Q000737:chemistry; D016718:Arachidonic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004952:Esters; D005998:Glycine / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D015289:Leukotrienes / Q000494:pharmacology; D019787:Linoleic Acid / Q000737:chemistry; D008041:Linoleic Acids / Q000737:chemistry; D008054:Lipid Peroxides / Q000494:pharmacology; D008084:Lipoxygenase / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008280:Magnetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012984:Software; D013025:Glycine max / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D017931:DNA Primers; D004952:Esters; D015289:Leukotrienes; D008041:Linoleic Acids; D008054:Lipid Peroxides; D016718:Arachidonic Acid; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; C054539:5,15-dihydroperoxyeicosatetraenoic acid; D006859:Hydrogen; D019787:Linoleic Acid; D007501:Iron; C101084:lipoxygenase L-1; D008084:Lipoxygenase; D000409:Alanine; D010100:Oxygen; D005998:Glycine