Hydrogen/Deuterium Exchange Study of Subtilisin Carlsberg During Prolonged Exposure to Organic Solvents
Hydrogen/Deuterium Exchange Study of Subtilisin Carlsberg During Prolonged Exposure to Organic Solvents
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DOI：
10.1002/bit.22147
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发表时间：
2009-03-01
影响因子：
3.8
通讯作者：
Barletta, Gabriel L.
中科院分区：
文献类型：
--
作者：
Fasoli, Ezio;Ferrer, Amaris;Barletta, Gabriel L.
以前有报道说，一种酶长期暴露于有机溶剂中会导致活性大幅下降。人们发现这种效应与催化剂的结构或它们在有机溶剂中的可能聚集无关，直到今天，活性丧失的原因仍不清楚。在本工作中，研究了嘉士伯枯草蛋白酶（SC）在长时间暴露于两种有机溶剂中的结构动力学，通过跟踪移动质子的氢/氘（H/D）交换。冻干后的酶在控制氘水活度的有机溶剂中孵育不同时间，并允许H/D交换发生。氢-2核磁共振（H-2 NMR）评估了氘交换量，从而使我们了解了模型酶在孵育期间和不同实验条件下的变化动态。我们的研究结果表明，SC在1,4-二氧六环（Diox）和乙腈（ACN）中长时间储存时，柔韧性下降，观察到蛋白质中快速交换质子（FEP）和慢交换质子（SEP）的表观交换速率常数(k)下降3- 10倍。我们的研究还表明，SC在ACN中比在Diox中更灵活（FEP和SEP在ACN中的k高3-20倍），表明酶动力学受到溶剂物理化学性质的影响。此外，酶的动力学也受到制备方法的影响：当酶用聚乙二醇（聚乙二醇化）进行化学修饰或用冠醚进行冻干时，观察到灵活性降低（FEP和SEP的k降低3- 10倍）。讨论了活性、对映体选择性(E)和结构动力学之间的可能关系，表明过去尝试的直接关联受到系统的多变量性质和复杂性的阻碍。
It has been previously reported that prolonged exposure of an enzyme to organic solvents leads to substantial decrease of activity. This effect was found to be unrelated to the catalysts' structure or their possible aggregation in organic solvents, and up to the present day the cause for activity loss remains unclear. In the present work, the structural dynamics of the serine protease subtilisin Carlsberg (SC) have been investigated during prolonged exposure to two organic solvents by following hydrogen/deuterium (H/D) exchange of mobile protons. The enzyme, after lyophilization, was incubated in organic solvents at controlled deuteriated water activity for different times and the H/D exchange was allowed to take place. The amount of deuterium exchanged was evaluated by H-2 NMR, which in turn gave us a Picture of the changing dynamics of our model enzyme during incubation and under different experimental conditions. Our results show that the flexibility of SC decreases during prolonged storage in 1,4-dioxane (Diox) and acetonitrile (ACN) as indicated by the observed 3- to 10-fold decrease in the apparent rate constants of exchange (k) of fast exchangeable protons (FEP) and slow exchangeable protons (SEP) in the protein. Our Study also shows that SC is more flexible in ACN than in Diox (k 3-20 times higher in ACN for the FEP and SEP), suggesting that enzyme dynamics are affected by solvent physicochemical properties. Additionally, the enzyme dynamics are also affected by the method of preparation: decreased flexibility (k decreases 3- to 10-fold for FEP and SEP) is observed when the enzyme is chemically modified with poly ethylene glycol (PEGylated) or colyophilized with crown ethers. A possible relationship between activity, enantioselectivity (E), and structural dynamics is discussed, demonstrating that direct correlations, as have been attempted in the past, are hampered by the multi-variable nature and complexity of the system.