Allosteric-Activation Mechanism of Bovine Chymosin Revealed by Bias-Exchange Metadynamics and Molecular Dynamics Simulations.
Allosteric-Activation Mechanism of Bovine Chymosin Revealed by Bias-Exchange Metadynamics and Molecular Dynamics Simulations.
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通过偏压交换元动力学和分子动力学模拟揭示牛凝乳酶的变构激活机制。
DOI：
10.1021/acs.jpcb.6b07491
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Ansari SM
中科院分区：
文献类型：
--
作者：
Ansari SM
天冬氨酸蛋白酶，牛凝乳酶，催化牛奶中κ-酪蛋白的蛋白质分解。牛凝乳酶−κ-酪蛋白复合体具有工业价值，因为该酶广泛应用于加工乳制品的生产中。该酶的载脂蛋白形式采用自抑制构象，其中Tyr77的侧链封闭结合部位。根据动力学、诱变和结晶学数据，已有广泛报道天然底物κ-酪蛋白的P8-P4残基中的HPHPH序列起变构激活剂的作用，但由于用实验方法研究这一过程存在挑战，其发生机制尚未被阐明。在这里，我们使用了两种计算技术，分子动力学和偏置交换元动力学模拟，研究了变构活化的机理，并计算了该过程的自由能面。模拟表明，变构激活是由κ-酪蛋白的HPHPH序列和凝乳酶蛋白的一个小的α-螺旋区域(残基112-116)相互作用启动的。α-螺旋中的一个小的构象变化导致Phe114的侧链腾出一个口袋，然后可能被Tyr77的侧链占据。κ-酪蛋白的HPHPH序列的存在显著改变了自抑制到开放转变的自由能表面。
The aspartic protease, bovine chymosin, catalyzes the proteolysis of κ-casein proteins in milk. The bovine chymosin−κ-casein complex is of industrial interest as the enzyme is used extensively in the manufacturing of processed dairy products. The apo form of the enzyme adopts a self-inhibited conformation in which the side chain of Tyr77 occludes the binding site. On the basis of kinetic, mutagenesis, and crystallographic data, it has been widely reported that a HPHPH sequence in the P8–P4 residues of the natural substrate κ-casein acts as the allosteric activator, but the mechanism by which this occurs has not previously been elucidated due to the challenges associated with studying this process by experimental methods. Here we have employed two computational techniques, molecular dynamics and bias-exchange metadynamics simulations, to study the mechanism of allosteric activation and to compute the free energy surface for the process. The simulations reveal that allosteric activation is initiated by interactions between the HPHPH sequence of κ-casein and a small α-helical region of chymosin (residues 112–116). A small conformational change in the α-helix causes the side chain of Phe114 to vacate a pocket that may then be occupied by the side chain of Tyr77. The free energy surface for the self-inhibited to open transition is significantly altered by the presence of the HPHPH sequence of κ-casein.
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