Biochemistry, University of Bayreuth, Universitätsstraße 30, BGI, 95447 Bayreuth, Germany.
Computational Biochemistry, University of Bayreuth, Universitätsstraße 30, BGI, 95447 Bayreuth, Germany.
J Phys Chem B. 2022 Jun 9;126(22):4035-4048. doi: 10.1021/acs.jpcb.2c01484. Epub 2022 May 24.
The catalytic mechanisms of serine and cysteine peptidases are similar: the proton of the nucleophile (serine or cysteine) is transferred to the catalytic histidine, and the nucleophile attacks the substrate for cleavage. However, they differ in an important aspect: cysteine peptidases form a stable ion-pair intermediate in a stepwise mechanism, while serine peptidases follow a concerted mechanism. While it is known that a positive electrostatic potential at the active site of cysteine peptidases stabilizes the cysteine anion in the ion-pair state, the physical basis of the concerted mechanism of serine peptidases is poorly understood. In this work, we use continuum electrostatic analysis and quantum mechanical/molecular mechanical (QM/MM) simulations to demonstrate that a destabilization of an anionic serine by a negative electrostatic potential in combination with a compact active site geometry facilitates a concerted mechanism in serine peptidases. Moreover, we show that an anionic serine would destabilize the protein significantly compared to an anionic cysteine in cysteine peptidases, which underlines the necessity of a concerted mechanism for serine peptidases. On the basis of our calculations on an inactive serine mutant of a natural cysteine peptidase, we show that the energy barrier for the catalytic mechanism can be substantially decreased by introducing a negative electrostatic potential and by reducing the relevant distances indicating that these parameters are essential for the activity of serine peptidases. Our work demonstrates that the concerted mechanism of serine peptidases represents an evolutionary innovative way to perform catalysis without the energetically expensive need to stabilize the anionic serine. In contrast in cysteine peptidases, the anionic cysteine is energetically easily accessible and it is a very efficient nucleophile, making these peptidases mechanistically simple. However, a cysteine is highly oxygen sensitive, which is problematic in an aerobic environment. On the basis of the analysis in this work, we suggest that serine peptidases represent an oxygen-insensitive alternative to cysteine peptidases.
亲核体（丝氨酸或半胱氨酸）的质子转移到催化组氨酸上，亲核体攻击底物进行裂解。然而，它们在一个重要方面有所不同：半胱氨酸肽酶在逐步机制中形成稳定的离子对中间体，而丝氨酸肽酶遵循协同机制。虽然已知半胱氨酸肽酶活性位点的正静电势稳定了离子对状态下的半胱氨酸阴离子，但丝氨酸肽酶协同机制的物理基础知之甚少。在这项工作中，我们使用连续静电分析和量子力学/分子力学（QM/MM）模拟来证明，在紧凑的活性位点几何形状下，负静电势对阴离子丝氨酸的去稳定作用有利于丝氨酸肽酶中的协同机制。此外，我们表明，与半胱氨酸肽酶中的阴离子半胱氨酸相比，阴离子丝氨酸会使蛋白质显著失稳，这突显了丝氨酸肽酶需要协同机制。基于我们对半胱氨酸肽酶天然活性位点丝氨酸突变体的计算，我们表明，通过引入负静电势和减小相关距离，可以显著降低催化机制的能垒，这表明这些参数对于丝氨酸肽酶的活性至关重要。我们的工作表明，丝氨酸肽酶的协同机制代表了一种无需昂贵能量来稳定阴离子丝氨酸即可进行催化的进化创新方式。相比之下，在半胱氨酸肽酶中，阴离子半胱氨酸很容易获得能量，并且是非常有效的亲核体，使得这些肽酶在机制上非常简单。然而，半胱氨酸对氧气非常敏感，这在有氧环境中是个问题。基于这项工作的分析，我们认为丝氨酸肽酶是对半胱氨酸肽酶的一种氧不敏感的替代选择。
