Laboratory of Bromatology, Faculty of Chemistry, Biochemistry and Pharmacy (FQByF), National University of San Luis, Chacabuco N° 917, San Luis, Argentina.
Institute of Applied Physics (INFAP) - Technological Scientific Center of San Luis - National Council of Scientific and Technique Research (CONICET), Ejército de los Andes N° 950, Block II, 2nd Floor, 5700, San Luis, Argentina.
Protein J. 2024 Jun;43(3):487-502. doi: 10.1007/s10930-024-10182-4. Epub 2024 Mar 7.
The present study aims at understanding the effect of organic solvents on the specific proteolytic activity and operational stability of asclepain cI in aqueous-organic media, using correlations between geometrical and structural parameters of asclepain cI. These correlations were determined by molecular dynamics (MD) simulations and the secondary structure of the enzyme validated by Fourier-transform Infrared (FTIR) spectroscopy. Asclepain cI exhibited significantly higher catalytic potential in 29 of the 42 aqueous-organic media tested, composed by 0.1 mM TRIS hydrochloride buffer pH 8 (TCB) and an organic solvent, than in buffer alone. Asclepain cI in water-organic miscible systems showed high FTIR spectral similarity with that obtained in TCB, while in immiscible systems the enzyme acquired different secondary structures than in buffer. Among the conditions studied, asclepain cI showed the highest catalytic potential in 50% v/v ethyl acetate in TCB. According to MD simulations, that medium elicited solvation and flexibility changes around the active center of asclepain cI and conducted to a new secondary structure with the active center preserved. These results provide valuable insights into the elucidation of the molecular mechanism of asclepain cI tolerance to organic solvents and pave the way for its future application for the synthesis of peptides in aqueous-organic media.
本研究旨在通过 Asclepain cI 的几何和结构参数之间的相关性，了解有机溶剂对其在水-有机介质中特定蛋白水解活性和操作稳定性的影响。这些相关性是通过分子动力学（MD）模拟和酶的二级结构（通过傅里叶变换红外（FTIR）光谱验证）来确定的。在测试的 42 种水-有机混合介质中的 29 种中，Asclepain cI 比在缓冲液中显示出显著更高的催化潜力，这些混合介质由 0.1 mM 的三羟甲基氨基甲烷盐酸缓冲液 pH 8（TCB）和有机溶剂组成。水-有机可混溶系统中的 Asclepain cI 与在 TCB 中获得的 FTIR 光谱具有高度相似性，而在不可混溶系统中，酶获得的二级结构与在缓冲液中不同。在所研究的条件中，Asclepain cI 在 TCB 中的 50% v/v 乙酸乙酯中表现出最高的催化潜力。根据 MD 模拟，该介质引起了 Asclepain cI 活性中心周围的溶剂化和灵活性变化，并导致活性中心保留的新二级结构。这些结果为阐明 Asclepain cI 对有机溶剂的耐受性的分子机制提供了有价值的见解，并为其未来在水-有机介质中合成肽的应用铺平了道路。
