Peptide hydrolysis in thermolysin: Ab initio QM/MM investigation of the Glu143-assisted water addition mechanism
Peptide hydrolysis in thermolysin: Ab initio QM/MM investigation of the Glu143-assisted water addition mechanism
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DOI：
10.1021/ct7000792
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发表时间：
2007-09-01
影响因子：
5.5
通讯作者：
Klein, Michael L.
中科院分区：
文献类型：
--
作者：
Blumberger, Jochen;Lamoureux, Guillaume;Klein, Michael L.
嗜热菌蛋白酶（Thermolysin，TLN）是研究最多的锌金属蛋白酶之一。然而，其作用机制仍在争论之中。为了研究目前最受欢迎的机制的能量可行性，我们对接了三肽的TLN的活性位点，并计算了自由能分布在量子力学/分子力学理论水平。该机制由三个不同的步骤组成：（i）Zn结合的水分子被Glu 143去质子化并攻击底物的羰基键;（ii）Glu 143将质子转移到酰胺氮原子;（iii）氮原子质子化，肽键不可逆地断裂。步骤i和步骤iii的自由能垒具有几乎相等的高度，分别为14.8和14.7千卡/摩尔，并且与对于类似衬底获得的有效实验活化垒12.1-13.6千卡/摩尔良好一致。亲核攻击的过渡态稳定化是通过在底物羰基氧原子和Zn离子之间形成弱配位键以及在底物和质子化His 231和两个溶剂分子之间形成三个强氢键来实现的。亲核攻击（步骤i）的过渡态比酶-底物复合物的结合更紧密，这意味着TLN符合鲍林关于过渡态稳定的假设。Glu 143，在第一次不利的酰胺氮原子的质子化取向，显示大的结构波动，促进重组的本地氢键网络和运输的质子1837离去基团的纳秒时间尺度上。目前的模拟进一步证明，Glu-143是一个非常有效的质子穿梭，应该被分配在TLN提出的任何反应机制的关键作用。
Thermolysin (TLN) is one of the best-studied zinc metalloproteases. Yet the mechanism of action is still under debate. In order to investigate the energetic feasibility of the currently most favored mechanism, we have docked a tripeptide to the active site of TLN and computed the free energy profile at the quantum mechanics/molecular mechanics level of theory. The mechanism consists of three distinct steps: (i) a Zn-bound water molecule is deprotonated by Glu143 and attacks the carbonyl bond of the substrate; (ii) Glu143 transfers the proton to the amide nitrogen atom; (iii) the nitrogen atom is protonated and the peptide bond is irreversibly broken. The free energy barriers for steps i and iii have almost equal heights, 14.8 and 14.7 kcal/mol, respectively, and are in good agreement with the effective experimental activation barrier obtained for similar substrates, 12.1-13.6 kcal/mol. Transition state stabilization for nucleophilic attack is achieved by formation of a weak coordination bond between the substrate carbonyl oxygen atom and the Zn ion and of three strong hydrogen bonds between the substrate and protonated His231 and two solvent molecules. The transition state for the nucleophilic attack (step i) is more tightly bonded than the enzyme-substrate complex, implying that TLN complies with Pauling's hypothesis regarding transition-state stabilization. Glu143, at first unfavorably oriented for protonation of the amide nitrogen atom, displayed large structural fluctuations that facilitated reorganization of the local hydrogen-bond network and transport of the proton to the 1837 leaving group on the nanosecond time scale. The present simulations give further evidence that Glu-143 is a highly effective proton shuttle which should be assigned a key role in any reaction mechanism proposed for TLN.