title: Locating a lipid at the portal to the lipoxygenase active site.
abstract: Lipoxygenase enzymes initiate diverse signaling pathways by specifically directing oxygen to different carbons of arachidonate and other polyunsaturated acyl chains, but structural origins of this specificity have remained unclear. We therefore determined the nature of the lipoxygenase interaction with the polar-end of a paramagnetic lipid by electron paramagnetic resonance spectroscopy. Distances between selected grid points on soybean seed lipoxygenase-1 (SBL1) and a lysolecithin spin-labeled on choline were measured by pulsed (electron) dipolar spectroscopy. The protein grid was designed by structure-based modeling so that five natural side chains were replaced with spin labels. Pairwise distances in 10 doubly spin-labeled mutants were examined by pulsed dipolar spectroscopy, and a fit to the model was optimized. Finally, experimental distances between the lysolecithin spin and each single spin site on SBL1 were also obtained. With these 15 distances, distance geometry localized the polar-end and the spin of the lysolecithin to the region between the two domains in the SBL1 structure, nearest to E236, K260, Q264, and Q544. Mutation of a nearby residue, E256A, relieved the high pH requirement for enzyme activity of SBL1 and allowed lipid binding at pH 7.2. This general approach could be used to locate other flexible molecules in macromolecular complexes.
authors.affiliation: Department of Biological Science Department, Florida State University, Tallahassee, FL, USA. gaffney@bio.fsu.edu
authors.forename: Betty J
authors.initials: BJ
authors.lastname: Gaffney
authors.forename: Miles D
authors.initials: MD
authors.lastname: Bradshaw
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Frausto
authors.forename: Fayi
authors.initials: F
authors.lastname: Wu
authors.forename: Jack H
authors.initials: JH
authors.lastname: Freed
authors.forename: Peter
authors.initials: P
authors.lastname: Borbat
mesh_terms: D020134:Catalytic Domain; D003497:Cyclic N-Oxides / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D054709:Lecithins / Q000737:chemistry / Q000378:metabolism; D008055:Lipids / Q000737:chemistry*; D008084:Lipoxygenase / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D012996:Solutions; D013025:Glycine max / Q000201:enzymology*; D013113:Spin Labels; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D003497:Cyclic N-Oxides; D054709:Lecithins; D008055:Lipids; D050505:Mutant Proteins; D012996:Solutions; D013113:Spin Labels; C017743:tempocholine; C101084:lipoxygenase L-1; D008084:Lipoxygenase