Insights from molecular dynamics simulations into pH-dependent enantioselective hydrolysis of ibuprofen esters by Candida rugosa lipase
Insights from molecular dynamics simulations into pH-dependent enantioselective hydrolysis of ibuprofen esters by Candida rugosa lipase
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DOI：
10.1093/protein/gzg135
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发表时间：
2003-12-01
期刊：
影响因子：
--
通讯作者：
Gautam, P
中科院分区：
文献类型：
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作者：
James, JJ;Lakshmi, BS;Gautam, P
在我们对念珠菌脂肪酶 (CRL) 水解布洛芬酯的持续研究中，发现了一个有趣的观察结果。 pH 在这些酯的立体定向水解中发挥着重要作用。 CRL 的瓣区在底物到达酶活性位点的过程中发挥着重要作用。在pH 5.6时，48%的布洛芬甲酯和5%的布洛芬丁酯在5.5小时内被水解，而在pH 7.2时，使用CRL在相同的反应时间内水解9%的布洛芬甲酯和45%的布洛芬丁酯。这使我们假设 CRL 在酸性 pH 下更喜欢布洛芬甲酯作为底物，在中性 pH 下更喜欢布洛芬丁酯作为底物。因此，为了了解 pH 在 CRL 对布洛芬酯的底物选择中的作用，我们使用 CRL (1CRL) 开放形式的晶体坐标，使用 GROMACS 在酸性和中性条件下进行 2 ns 的分子动力学 (MD) 模拟。将在酸性和中性条件下模拟后获得的最终结构与能量最小化结构进行比较，并计算均方根偏差（r. m. s. ds）。河。米。 s。 d.发现 CRL 皮瓣在中性 pH 值下的收缩力大于 CRL 皮瓣在酸性 pH 值下的收缩力。瓣片在中性 pH 值下打开的程度允许体积较大的底物（布洛芬丁酯）扩散到活性位点，并提供适合该特定底物的最佳酶-底物。在酸性 pH 值下，瓣片的开口减小，从而容纳更紧凑的底物，即布洛芬甲酯。因此，使用MD的模拟实验为CRL在水性环境中的pH依赖性底物选择性提供了合理的见解。
An interesting observation was found during our continued studies on the hydrolysis of ibuprofen esters by Candida rugosa lipase (CRL). An important role is played by pH in the stereospecific hydrolysis of these esters. The flap region of CRL plays a significant role in the access of the substrate to the active site of the enzyme. At pH 5.6, 48% of the methyl ester and 5% of the butyl ester of ibuprofen were hydrolysed in 5.5 h, whereas at pH 7.2, 9% of methyl ester and 45% of the butyl ester of ibuprofen was hydrolysed in a identical reaction time using CRL. This lead us to assume that CRL prefers the methyl ester of ibuprofen as a substrate at an acidic pH and the butyl ester of ibuprofen at a neutral pH. Therefore, in order to understand the role of pH in the substrate selection by CRL for the esters of ibuprofen we used the crystallographic coordinates of the open form of the CRL ( 1CRL) for molecular dynamics ( MD) simulations under acidic and neutral conditions for 2 ns using GROMACS. The final structures obtained after simulation in acidic and neutral conditions were compared with the energy-minimized structure, and the root-mean-square deviations (r. m. s. ds) were calculated. The r. m. s. d. of the CRL flap at neutral pH was found to be greater than that of the CRL flap at acidic pH. The extent to which the flap opens at neutral pH allowed the bulkier substrate, the butyl ester of ibuprofen, to diffuse into the active site and provides the best enzyme - substrate fit for this specific substrate. At acidic pH there is a decreased opening of the flap thereby accommodating a more compact substrate, namely the methyl ester of ibuprofen. Thus, simulation experiments using MD provide reasonable insight for the pH-dependent substrate selectivity of CRL in aqueous environments.