Interactions of Lipoxygenase with Paramagnetic Substrate Analogs

Abstract

How fatty acid substrates and inhibitors enter and reside in the internal cavity of lipoxygenases is still unresolved. A medium-resolution, solution structure of lipoxygenase is obtained by positioning spin labels on a grid of five sites near the protein surface, all 20-30 A distant from iron. Cysteine substitutions, for subsequent spin labeling, are made on a background of soybean lipoxygenase in which serine replaces four native cysteines. Dynamics and solvent exposure of the spin labeled side chains are characterized by solution electron paramagnetic resonance (EPR) spectra in the presence and absence of viscogens and fatty acid substrate analogs. Additionally, more detailed structural information is obtained from site directed spin labeling of helix-2 to examine how binding of inhibitors influences the conformation of this critical helix. Examples of changes in separation of outer hyperfine extrema in the EPR spectra when 30% sucrose is added include the following. For helix-2: spin labeled mutants T259C (change=0.17mT); K260C (mobile); F270C (0.11mT); and for grid points: L480C (0.16mT); L541C (0.16mT); and A569C (mobile). This study precedes determining the location of paramagnetic substrate analogs by examining dipolar-determined distances between the protein labeled sites and spin labeled substrate analogs.View Large Image | View Hi-Res Image | Download PowerPoint Slide