1H-NMR spectroscopic study on the binding of alcohols to soybean lipoxygenase-1

Abstract

The line-shape of the EPR signal around g 6 of yellow lipoxygenase-1, obtained upon addition of I molar equivalent of 13- l s- hydroperoxy-9-cis ,11- trans-octadecadienoic acid to the native enzyme (linoleate:oxygen oxidoreductase, EC 1.13.11.12), is strongly affected by alcohols. NMR spectra of solutions of alcohols to which lipoxygenase has been added show a line-broadening of the proton resonances which is due to proton relaxation enhancement from magnetic interaction between iron and protons. This can be taken as direct evidence for binding of alcohols in the vicinity of iron. For unbranched alcohols the line-broadening gradually increases, going from the methyl protons to the protons on carbon atom 1, indicating that the latter are closer to iron. Titrations of yellow lipoxygenase with ethanol, 1-butanol and 1-hexanol reveal that the affinity of the alcohols increases with longer carbon chain length; their binding constants were found to be 260, 30 and approx. 3 mM respectively. The distances between protons of bound alcohol and iron were calculated with the Solomon-Bloembergen equation, leading to values of approx. 6 Å, for the distance between iron and the methyl protons. A hydrophobic binding of the alcohols to the enzyme is proposed in line with the mode of binding of the natural substrates, polyunsaturated fatty acids.