Structural origins of the interfacial activation in Thermomyces (Humicola)lanuginosa lipase

The already known X-ray structures of lipases provide little evidence about initial, discrete structural steps occurring in the first phases of their activation in the presence of lipids (process referred to as interfacial ac tivation). To address this problem, five new Thermomyces (formerly Humicola ) lanuginosa lipase (TIL) crystal structures have been solved and compared with four previously reported structures of this enzyme. The bias coming fr om different crystallization media has been minimized by the growth of all crystals under the same crystallization conditions, in the presence of dete rgent/lipid analogues, with low or high ionic strength as the only main var iable. Resulting structures and their characteristic features allowed the i dentification of three structurally distinct species of this enzyme: low ac tivity form (LA), activated form (A), and fully Active (FA) form. The isome rization of the Cys268-Cys22 disulfide, synchronized with the formation of a new, short alpha (0) helix and flipping of the Arg84 (Arginine switch) lo cated in the lid's proximal hinge, have been postulated as the key, structu ral factors of the initial transitions between LA and A forms. The experime ntal results were supplemented by theoretical calculations. The magnitude o f the activation barrier between LA (ground state) and A (end state) forms of TIL (10.6 kcal/mol) is comparable to the enthalpic barriers typical for ring flips and disulfide isomerizations at ambient temperatures. This sugge sts that the sequence of the structural changes, as exemplified in various TlL crystal structures, mirror those that may occur during interfacial acti vation.