Dynamic Behavior of Fatty Acid Spin Labels within a Binding Site of Soybean Lipoxygenase-1

Abstract

The putative substrate-binding site in lipoxygenases is long and internal. There is little direct evidence about how the unsaturated fatty acid substrates enter and move within the cavity in order to position correctly for electron transfer reactions with the catalytic non-heme iron. An EPR spectroscopy approach, with spin labeled fatty acids, is taken here to investigate dynamic behavior of fatty acids bound to soybean lipoxygenase-1. The probes are labeled on carbons 5-, 8-, 10-, 12-and 16-of stearic acid. The EPR-determined affinity for the enzyme increases as the length of the alkyl end of the probe increases, with ΔΔG of −190 cal/methylene. The probes in the series exhibit similar enhanced paramagnetic relaxation by the iron center. These results indicate that the members of the series have a common binding site. All of the bound probes undergo considerable local mobility. The stearate spin labeled on carbon 5 has highest affinity for the lipoxygenase and it is a competitive inhibitor, with K i 9 μM. Surprisingly, this stearate labeled near the carboxyl end undergoes more local motion than those labeled in the middle of the chain, when it is bound. This shows that the carboxyl end of the fatty acid spin label is not rigidly docked on the protein. During catalysis, repositioning of the substrate carboxyl on the protein surface may be coupled to motion of portions of the chain undergoing reaction. KeywordsLipoxygenase; spin label; fatty acid; substrate motion; paramagnetic protein Lipoxygenase (LOX) enzymes catalyze the first step in one of the major pathways to molecules derived from arachidonic acid. A theme emerging from the medical perspective on different human lipoxygenases is one of positive and negative influences on human health from these molecules (1,2). Thus, there is much interest in being able to pharmaceutically modulate the activity of selected members of the lipoxygenase family while leaving other members functioning normally (3,4). Indeed, inhibitors selective for 5-, 12-or 15-arachidonic acid lipoxygenases have been developed (5-7). Lipoxygenases from both plant and animal sources have overall structural similarity with a long internal cavity that passes by the catalytic iron ion, based on available X-ray structures (8)(9)(10)(11)(12)(13)(14). Some determinants of substrate binding within this cavity have been examined by mutation and modeling (15)(16)(17)(18)(19)(20), but there is little direct * To whom correspondence should be addressed: Tel. (850) 644-8547. Fax: (850) 644-0481. E-mail: gaffney@bio.fsu.edu. 1 ABBREVIATIONS: cmc, critical micelle concentration; EPR, electron paramagnetic resonance; LOX, any lipoxygenase; SBL1, soybean lipoxygenase isoform-1; 13S-HPODE, 13S-hydroperoxyoctadecadienoic acid; cAOS, coral allene oxide synthase; doxyl-, 1-oxyl-2,2,5,5-tetramethyloxazolidinyl-; x-DSA, a doxyl stearic acid spin label with doxyl ring on carbon x; TEMPOL, 1-oxyl-2,2,6,6-tetramethyl-4-piperidinol; 2A zz , rigid limit separation of outer extrema in spin label EPR spectra; 2A ...

Solid-state NMR paramagnetic relaxation enhancement immersion depth studies in phospholipid bilayers

“…It has been shown that correlation times vary with the positions in DOXYL-labeled stearic acid (6.5 ns for 5-SDA, 12.4 ns for 10-SDA) [61]. We used an estimated correlation time τ c in calculating the distance from the PRE data of 1 ns.…”

EPR Spectroscopic Studies of Lipoxygenases

“…For instance,t he spin labeled stearic acid derivative 8-DSA,w ith nine methylenes on the terminal methyle nd, has 31 mm affinity in solution. [50]…”

Lipoxygenases and plant cell metabolism regulation

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