ABSTRACT
Incubation of fluorescein 5'-isothiocyanate (FITC) with soybean lipoxygenase produces the coupling of 1 mol of fluorophore to 1 mol of enzyme. Derivatized lipoxygenase lost 40% activity through a mixed-type inhibitory mechanism. The quenching by IK of the fluorescence of FITC-labeled lipoxygenase shows that the fluorophore is located near the surface of the protein. The partial impediment of the FITC labeling when the substrate is present in the medium, together with data of the tryptic digestion, suggests that FITC is attached via the access channel of substrate to the catalytic site. Labeling does not induce appreciable modification of the enzyme specificity, suggesting that the position of substrate in the active site is not modified by the labeling. The results obtained strongly suggest that FITC labels soybean lipoxygenase specifically at a lysyl residue which contributes to fixation of the carboxylic end of the substrate to the active center. The experimental data obtained from the quenching of FITC fluorescence by NDGA reveal that this molecule interacts with the protein near the catalytic site.
Subject(s)
Fluorescein-5-isothiocyanate , Fluorescent Dyes , Glycine max/enzymology , Lipoxygenase/chemistry , Lipoxygenase/metabolism , Binding Sites , Catalytic Domain , Fluorescence Polarization , Kinetics , Lipoxygenase Inhibitors/pharmacology , Lysine/chemistry , Masoprocol/pharmacology , Substrate SpecificityABSTRACT
In this study, lipoxygenase from potato tuber has been purified by a method involving hydrophobic chromatography and the purified enzyme immobilized by covalent coupling to oxirane acrylic beads. The immobilized lipoxygenase exhibited increased long-term stability without a significant modification of the kinetic parameters. The comparative study on the effects of inhibitors such as dithizone, NDGA, phenidone, and beta-mercaptoethanol on the free and immobilized enzyme highlighted the importance of the lipoxygenase--support interaction, concluding that the immobilization process could cause the protection of the iron atom in the enzyme. The enzymatic specificity was maintained for the immobilized lipoxygenase, and their stability increased as compared to the free enzyme, making if feasible to use the enzyme in a multistep reaction to produce large quantities of leukotriene A4 or other related compounds of interest in the chemical industry and medicine.
