pH-induced domain interaction and conformational transitions of lipoxygenase-1

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 ir reversible conformation with the properties of molten globule state with a mid point of transition at pH 2.4. This intermediate state lost iron irreve rsibly. In alkaline pH at 11.5, LOX1 underwent partial unfolding with the e xposure of cysteine residues with subsequent oxidation of a pair of cystein e residues in the C-terminal domain and this intermediate showed some prope rties of molten globule state and retained 35% of activity. Beyond pH 12.0, the enzyme was completely inactivated irreversibly due to irreversible con formational changes. The pH-dependent urea-induced unfolding of LOX1 sugges ted that LOX1 was more stable at pH 7.0 and least stable at pH 9.0. Further more, the urea-induced unfolding of LOX1 indicated that the unfolding was b iphasic due to pH-dependent domain interactions and involved sequential unf olding of domains. The loss of enzyme activity at pH 4.0 and 7.0 occurred m uch earlier to unfolding of the C-domain at all pHs studied. The combinatio n of urea-induced unfolding measurements and limited proteolysis experiment s suggested that at pH 4.0, the domains in LOX1 were less interactive and e xisted as tightly folded units. Furthermore, these results confirmed the co ntribution of ionic interactions in the interdomain contacts. (C) 2000 Else vier Science B.V. All rights reserved.