The Enzymatic Activity of Lipases Correlates with Polarity-Induced Conformational Changes: A Trp-Induced Quenching Fluorescence Study
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- 作者：Jakob Skjold-J酶rgensen ; Vikram K. Bhatia ; Jesper Vind ; Allan Svendsen ; Morten J. Bjerrum ; David Farrens
- 刊名：Biochemistry
- 出版年：2015
- 出版时间：July 14, 2015
- 年：2015
- 卷：54
- 期：27
- 页码：4186-4196
- 全文大小：538K
- ISSN：1520-4995
文摘
Triacylglycerol hydrolases (EC 3.1.1.3) are thought to become activated when they encounter the water鈥搇ipid interface causing a 鈥渓id鈥?region to move and expose the catalytic site. Here, we tested this idea by looking for lid movements in Thermomyces lanuginosus lipase (TL lipase), and in variants with a mutated lid region of esterase (Esterase) and esterase/lipase (Hybrid) character. To measure lid movements, we employed the tryptophan-induced quenching (TrIQ) fluorescence method to measure how effectively a Trp residue on the lid of these mutants (at position 87 or 89) could quench a fluorescent probe (bimane) placed at nearby site 255 on the protein. To test if lid movement is induced when the enzyme detects a lower-polarity environment (such as at the water鈥搇ipid interface), we performed these studies in solvents with different dielectric constants (蔚). The results show that lid movement is highly dependent on the particular lid residue composition and solvent polarity. The data suggest that in aqueous solution (蔚 = 80), the Esterase lid is in an 鈥渙pen鈥?conformation, whereas for the TL lipase and Hybrid, the lid remains 鈥渃losed鈥? At lower solvent polarities (蔚 < 46), the lid region for all of the mutants is more 鈥渙pen鈥? Interestingly, these behaviors mirror the structural changes thought to take place upon activation of the enzyme at the water鈥搇ipid interface. Together, these results support the idea that lipases are more active in low-polarity solvents because the lid adopts an 鈥渙pen鈥?conformation and indicate that relatively small conformational changes in the lid region play a key role in the activation mechanism of these enzymes.