Skip to Content
Merck
  • Dynamic behavior of fatty acid spin labels within a binding site of soybean lipoxygenase-1.

Dynamic behavior of fatty acid spin labels within a binding site of soybean lipoxygenase-1.

Biochemistry (2006-10-13)
Fayi Wu, Betty J Gaffney
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 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.

MATERIALS
Product Number
Brand
Product Description

Avanti
16:0-10 Doxyl PC, Avanti Research - A Croda Brand 810603C
Avanti
16:0-5 Doxyl PC, Avanti Research - A Croda Brand 810601P, powder
Avanti
16:0-12 Doxyl PC, Avanti Research - A Croda Brand 810600P, powder
Avanti
16:0-5 Doxyl PC, Avanti Research - A Croda Brand 810601C
Avanti
16:0-10 Doxyl PC, Avanti Research - A Croda Brand 810603P, powder
Avanti
16:0-16 Doxyl PC, Avanti Research - A Croda Brand 810604C
Avanti
16:0-16 Doxyl PC, Avanti Research - A Croda Brand 810604P, powder
Avanti
16:0-7 Doxyl PC, Avanti Research - A Croda Brand 810602P, powder