Lipoxygenase isoenzymes: A spectroscopic and structural characterization of soybean seed enzymes

Abstract

Applying recent developments in protein purification techniques, a number of lipoxygenase isoenzymes have been isolated in satisfactory quantities for a detailed physical and structural characterization. Four seed isoenzymes from two soybean cultivars have been studied by peptide mapping, free thiol and iron content determinations, and C-terminal analysis as well as by uv-visible absorption and EPR spectroscopy. While differences between the type 1 enzyme and the other isoenzymes were readily detected using proteolytic peptide mapping, digestion with dilute hydrochloric acid and C-terminal analysis both revealed structural features which were similar in all of the isoenzymes. One clear difference between the lipoxygenases was in their free sulfhydryl group content. The uv-visible absorption spectrum of each native isoenzyme was consistent with expectations for the experimental aromatic amino acid content. All of the isoenzymes contained one non-heme iron atom per molecule of protein. The oxidation of each isoenzyme with product hydroperoxide resulted in the conversion of the iron from an EPR silent state into several forms with EPR signals characteristic of high spin iron(III). The EPR spectra of all isoenzymes were remarkably similar. A time course EPR and catalytic activity study revealed that the various EPR active states represent a complex equilibrium between iron atoms in different environments. The pH dependence for the EPR and absorption spectroscopy lends support to the hypothesis that acid/base chemistry represents an important aspect of lipoxygenase catalysis.

Iron Extraction from Soybean Lipoxygenase 3 and Reconstitution of Catalytic Activity from the Apoenzyme

“…The iron(III) form of lipoxygenase 3 has distinctive spectroscopic features (14). For example, EPR spectra of samples of the product oxidized form of the enzyme at pH 8.0 contain signals at g6 and g4.3.…”

“…Iron concentration determinations were carried out by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) at 259.9 nm) using a 1000 ppm Certified Atomic Absorption Standard (Fisher Scientific) solution for calibration. EPR spectroscopy was conducted on frozen solutions of the protein treated with (9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid (1.1X) as previously described (14). The 9 GHz measurements were performed using a Varian Century Line Spectrometer equipped with a home-made, gas phase liquid helium transfer line and quartz dewar cavity insert operating at 25 K.…”

Lipoxygenase-1 Inhibition with a Series of Half-Product Analogs

“…Provar (26). The concentration of the enzyme was determined from the absorbance at 280 nm using a molar extinction coefficient of 120,000 liters mol Ϫ1 cm Ϫ1 (27). Linoleic acid was obtained from Sigma (St. Louis, MO).…”

Shape and Specificity in Mammalian 15-Lipoxygenase Active Site

“…They are widely distributed in plants and animals (2,3) and contain 1 mol of non-heme iron per mol of enzyme (4,5) which is essential for the oxygenase reaction (6). According to the currently used nomenclature, LOs are categorized with respect to their positional specificity of arachidonic acid oxygenation such that they are referred to as 5-, 8-, 11-, 12-, and 15-LOs.…”