title: Electrospray ionization mass spectrometry of soybean lipoxygenases: N-terminal acetylation, chemical modification, and solution conformation.
abstract: Electrospray ionization mass spectrometry was used to examine both the covalent structure and solution conformation of the soybean lipoxygenases. The post-translational modifications of two lipoxgyenases were identified as N-terminal acetylations by tandem mass spectrometry of peptides generated by trypsin digestion. The N-terminal sequence suggests that the proteins were substrates for the plant homolog of the N-terminal acetyltransferase complex C in yeast. Analysis of samples of native lipoxygenase-3 produced ions corresponding within experimental error to the mass of the N-acetylated polypeptide and one iron atom. The precision of the measurements was within roughly 100 ppm for the 96,856 Da protein. This made it possible to detect the addition of a single oxygen atom to the enzyme in a chemical modification reaction with cumene hydroperoxide. The acid-induced denaturation of lipoxygenase-3, which was accompanied by nearly complete loss of catalytic activity, was observed below pH 3.5 with the appearance of ions in the mass spectrum derived from the apoprotein. There was no evidence for the loss of iron in the absence of unfolding. Solutions of lipoxygenase-3 incubated in 0.1M acetic acid produced ions with a novel charge state distribution suggesting a unique conformation. Circular dichroism measurements showed that the secondary structure features of the native protein were retained in the new conformation. Dynamic light scattering revealed that the new conformation was not a consequence of protein aggregation as the hydrodynamic radius of lipoxygenase-3 was significantly smaller in acetic acid solution than at pH 7.0. Remarkably, the enzyme incubated in acetic acid retained full catalytic activity.
authors: Department of Chemistry, University of Toledo, Toledo, Ohio 43606, USA.
authors: Amber M
authors: AM
authors: Peariso
authors: Kellie M
authors: KM
authors: Nicholson
authors: R
authors: R
authors: Benjamin Jones
authors: Kari B
authors: KB
authors: Green-Church
authors: Max O
authors: MO
authors: Funk
mesh_terms: D000107:Acetylation; D006863:Hydrogen-Ion Concentration; D008084:Lipoxygenase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D013025:Glycine max / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D010940:Plant Proteins; C095864:lipoxygenase 3; D008084:Lipoxygenase