Structural Origins of the Interfacial Activation in Thermomyces (Humicola) lanuginosa Lipase

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• 作者：A. Marek Brzozowski ; Hugh Savage ; Chandra S. Verma ; Johan P. Turkenburg ; David M. Lawson ; Allan Svendsen ; and Sham Patkar
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：December 12, 2000
• 年：2000
• 卷：39
• 期：49
• 页码：15071 - 15082
• 全文大小：1047K
• 年卷期：v.39,no.49(December 12, 2000)
• ISSN：1520-4995

文摘

The already known X-ray structures of lipases provide little evidence about initial, discretestructural steps occurring in the first phases of their activation in the presence of lipids (process referredto as interfacial activation). To address this problem, five new Thermomyces (formerly Humicola)lanuginosa lipase (TlL) crystal structures have been solved and compared with four previously reportedstructures of this enzyme. The bias coming from different crystallization media has been minimized bythe growth of all crystals under the same crystallization conditions, in the presence of detergent/lipidanalogues, with low or high ionic strength as the only main variable. Resulting structures and theircharacteristic features allowed the identification of three structurally distinct species of this enzyme: lowactivity form (LA), activated form (A), and fully Active (FA) form. The isomerization of the Cys268-Cys22 disulfide, synchronized with the formation of a new, short ₀ helix and flipping of the Arg84(Arginine switch) located in the lid's proximal hinge, have been postulated as the key, structural factorsof the initial transitions between LA and A forms. The experimental results were supplemented by theoreticalcalculations. The magnitude of the activation barrier between LA (ground state) and A (end state) formsof TlL (10.6 kcal/mol) is comparable to the enthalpic barriers typical for ring flips and disulfideisomerizations at ambient temperatures. This suggests that the sequence of the structural changes, asexemplified in various TlL crystal structures, mirror those that may occur during interfacial activation.