title: Comparison of conformational changes and inactivation of soybean lipoxygenase-1 during urea denaturation.
abstract: The unfolding and inactivation of soybean lipoxygenase-1 during urea denaturation has been compared. Equilibrium study indicates that inactivation of the enzyme occurs at low urea concentrations before significant conformational change of the molecule as a whole. In the presence of 6.0 M urea, the unfolding of soybean lipoxygenase-1, as monitored by fluorescence intensity, is a triphasic process, while the inactivation of the enzyme shows single-phase kinetics. The rate constant of inactivation is consistent with that of the fast conformational change of the enzyme. The results suggest that active sites of lipoxygenase-1 containing iron cofactor are situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole. The kinetic theory of substrate reactions catalyzed by unstable enzymes (Duggleby (1986) J. Theor. Biol. 123, 67-80) has been applied to study the effect of substrate on enzyme inactivation. On the basis of the kinetic equation of substrate reaction in the presence of urea, inactivation rate constants for the free enzyme and enzyme-substrate complex have been determined. The substrate, linoleic acid, has no effect on inactivation of the ferric form of lipoxygenase-1.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, People's Republic of China.
authors.forename: Y
authors.initials: Y
authors.lastname: Wu
authors.forename: Z X
authors.initials: ZX
authors.lastname: Wang
mesh_terms: D001665:Binding Sites / Q000502:physiology; D004795:Enzyme Stability / Q000187:drug effects; D005453:Fluorescence; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013025:Glycine max / Q000201:enzymology*; D014508:Urea / Q000494:pharmacology
chemical_list: D010940:Plant Proteins; D014508:Urea; D007501:Iron; D008084:Lipoxygenase