Lipoxygenase isoenzymes: a spectroscopic and structural characterization of soybean seed enzymes.
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.
Draheim
J E
JE
Department of Chemistry, University of Toledo, Ohio 43606.
Carroll
R T
RT
McNemar
T B
TB
Dunham
W R
WR
Sands
R H
RH
Funk
M O
MO
D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008084:Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000201:enzymology*; D013025:Glycine max / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D010940:Plant Proteins; D008084:Lipoxygenase