title: pH-induced domain interaction and conformational transitions of lipoxygenase-1.
abstract: The multidomain structure of soybean LOX1 was examined over the pH range 1-12. Lipoxygenase-1 activity was reversible over broad pH range of 4-10 due to the reversibility of conformational states of the molecule. Below pH 4.0, due to collapse in hydrophobic interactions, the enzyme unfolded to an irreversible conformation with the properties of molten globule state with a mid point of transition at pH 2.4. This intermediate state lost iron irreversibly. In alkaline pH at 11.5, LOX1 underwent partial unfolding with the exposure of cysteine residues with subsequent oxidation of a pair of cysteine residues in the C-terminal domain and this intermediate showed some properties of molten globule state and retained 35% of activity. Beyond pH 12.0, the enzyme was completely inactivated irreversibly due to irreversible conformational changes. The pH-dependent urea-induced unfolding of LOX1 suggested that LOX1 was more stable at pH 7.0 and least stable at pH 9.0. Furthermore, the urea-induced unfolding of LOX1 indicated that the unfolding was biphasic due to pH-dependent domain interactions and involved sequential unfolding of domains. The loss of enzyme activity at pH 4. 0 and 7.0 occurred much earlier to unfolding of the C-domain at all pHs studied. The combination of urea-induced unfolding measurements and limited proteolysis experiments suggested that at pH 4.0, the domains in LOX1 were less interactive and existed as tightly folded units. Furthermore, these results confirmed the contribution of ionic interactions in the interdomain contacts.
authors.affiliation: Department of Protein Chemistry and Technology, Central Food Technological Research Institute, 570 013, Mysore, India.
authors.forename: E
authors.initials: E
authors.lastname: Sudharshan
authors.forename: S
authors.initials: S
authors.lastname: Srinivasulu
authors.forename: A G
authors.initials: AG
authors.lastname: Appu Rao
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007501:Iron / Q000032:analysis; D008084:Lipoxygenase / Q000737:chemistry / Q000378:metabolism*; D010434:Pepsin A / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D013025:Glycine max / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014508:Urea / Q000737:chemistry
chemical_list: D000817:Anilino Naphthalenesulfonates; D013438:Sulfhydryl Compounds; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D007501:Iron; D008084:Lipoxygenase; D010434:Pepsin A