Rigidifying a De Novo Enzyme Increases Activity and Induces a Negative Activation Heat Capacity.
Rigidifying a De Novo Enzyme Increases Activity and Induces a Negative Activation Heat Capacity.
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DOI：
10.1021/acscatal.1c01776
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发表时间：
2021-09-17
期刊：
影响因子：
12.9
通讯作者：
Anderson JLR
中科院分区：
文献类型：
--
作者：
Hindson SA;Bunzel HA;Frank B;Svistunenko DA;Williams C;van der Kamp MW;Mulholland AJ;Pudney CR;Anderson JLR
构象取样深刻地影响酶的整体活性和温度依赖性。过氧化物酶由于其广泛的潜在生物转化而成为高价值生物催化的通用平台。在这里，我们探讨的作用，构象采样介导的从头过氧化物酶C45的活动。我们证明，2，2，2-三氟乙醇（TFE）影响酶的构象状态的平衡，趋向于一个更全面的刚性结构。这与稳定性和活性的增加相关。值得注意的是，这些影响是伴随着出现的曲率的温度-活性曲线，权衡活动增益在环境温度下的高温损失。我们应用大分子速率理论（MMRT）来理解酶的温度依赖性数据。这些数据表明蛋白质刚性的增加与蛋白质动力学在基态和过渡态之间的分布差异有关。我们比较从头酶活性的热力学的天然过氧化物酶，辣根过氧化物酶。我们发现，本地酶类似于硬化的从头酶的酶催化的热力学和蛋白质动力学之间的基态和过渡态的假定分布。TFE的加入显然使C45的行为更像天然酶。我们的数据表明，通过操纵蛋白质刚性来提高生物催化活性的稳健的通用策略;特别是对于功能性从头蛋白质催化剂，这可以提供更多的酶样催化剂，而无需进一步的理性工程，计算重新设计或定向进化。
Conformational sampling profoundly impacts the overall activity and temperature dependence of enzymes. Peroxidases have emerged as versatile platforms for high-value biocatalysis owing to their broad palette of potential biotransformations. Here, we explore the role of conformational sampling in mediating activity in the de novo peroxidase C45. We demonstrate that 2,2,2-triflouoroethanol (TFE) affects the equilibrium of enzyme conformational states, tending toward a more globally rigid structure. This is correlated with increases in both stability and activity. Notably, these effects are concomitant with the emergence of curvature in the temperature-activity profile, trading off activity gains at ambient temperature with losses at high temperatures. We apply macromolecular rate theory (MMRT) to understand enzyme temperature dependence data. These data point to an increase in protein rigidity associated with a difference in the distribution of protein dynamics between the ground and transition states. We compare the thermodynamics of the de novo enzyme activity to those of a natural peroxidase, horseradish peroxidase. We find that the native enzyme resembles the rigidified de novo enzyme in terms of the thermodynamics of enzyme catalysis and the putative distribution of protein dynamics between the ground and transition states. The addition of TFE apparently causes C45 to behave more like the natural enzyme. Our data suggest robust, generic strategies for improving biocatalytic activity by manipulating protein rigidity; for functional de novo protein catalysts in particular, this can provide more enzyme-like catalysts without further rational engineering, computational redesign, or directed evolution.
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DOI：
10.1039/c8cc02426j
发表时间：
2018-06-19
期刊：
Chemical communications (Cambridge, England)
影响因子：
--
作者：
Maria-Solano MA ;Serrano-Hervás E ;Romero-Rivera A ;Iglesias-Fernández J ;Osuna S
通讯作者：
Osuna S
DOI：
10.1038/nsb1297-1032
发表时间：
1997-12-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Gajhede, M;Schuller, DJ;Poulos, TL
通讯作者：
Poulos, TL
影响因子：
8
作者：
Go, Abigail;Kim, Seho;Hecht, Michael H.
通讯作者：
Hecht, Michael H.
影响因子：
4.3
作者：
Candotti, Michela;Orozco, Modesto
通讯作者：
Orozco, Modesto
影响因子：
5.5
作者：
Frank SA
通讯作者：
Frank SA