Dynamic behavior of fatty acid spin labels within a binding site of soybean lipoxygenase-1.
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 to position themselves 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 C5, C8, C10, C12, and C16 of stearic acid. The EPR-determined affinity for the enzyme increases as the length of the alkyl end of the probe increases, with a DeltaDeltaG 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 at C5 has the highest affinity for the lipoxygenase, and it is a competitive inhibitor, with a K(i) of 9 muM. 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.
Wu
Fayi
F
Department of Biological Science, Florida State University, Tallahassee, Florida 32306-4370, USA.
Gaffney
Betty J
BJ
D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D005227:Fatty Acids / Q000378:metabolism*; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D013025:Glycine max / Q000201:enzymology*; D013113:Spin Labels; D013229:Stearic Acids / Q000378:metabolism; D013816:Thermodynamics
D005227:Fatty Acids; D013113:Spin Labels; D013229:Stearic Acids; D008084:Lipoxygenase