Tryptophan 500 and arginine 707 define product and substrate active site binding in soybean lipoxygenase-1.
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 pi-pi 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.
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
Viola C
VC
Ruddat
Rakesh
R
Mogul
Ilya
I
Chorny
Cameron
C
Chen
Noah
N
Perrin
Stephanie
S
Whitman
Victor
V
Kenyon
Matthew P
MP
Jacobson
Claude F
CF
Bernasconi
Theodore R
TR
Holman
D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D019295:Computational Biology / Q000379:methods; D003198:Computer Simulation; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation; D007700:Kinetics; D015289:Leukotrienes / Q000737:chemistry; D008024:Ligands; D008041:Linoleic Acids / Q000737:chemistry; D008054:Lipid Peroxides / Q000737:chemistry; D008084:Lipoxygenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D013025:Glycine max / Q000201:enzymology*; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry*
D015289:Leukotrienes; D008024:Ligands; D008041:Linoleic Acids; D008054:Lipid Peroxides; C038649:13-hydroperoxy-9,11-octadecadienoic acid; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; D014364:Tryptophan; D001120:Arginine; D008084:Lipoxygenase