Contribution of Oxyanion Stabilization to Kemp Eliminase Efficiency
Contribution of Oxyanion Stabilization to Kemp Eliminase Efficiency
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DOI：
10.1021/acscatal.0c00575
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发表时间：
2020-04-17
期刊：
影响因子：
12.9
通讯作者：
Hilvert, Donald
中科院分区：
文献类型：
--
作者：
Kries, Hajo;Bloch, Joel S.;Hilvert, Donald
生物学和生物催化中的重要反应涉及从碳中提取质子。当产生的阴离子电荷从碳原子离域到氧原子时，这些去质子化可以通过氧阴离子稳定催化加速。谷氨酰胺侧链（Gln50）的氧阴离子稳定被认为加速了HG3.17中C-H质子的提取，HG3.17是一种计算设计的生物催化剂，经过进化优化，具有类似酶的效率。我们提供了六个Gln50突变体的原子分辨率动力学数据和晶体结构，表明氢键供体谷氨酰胺比“油脂”蛋氨酸具有惊人的小优势。然而，紧密排列的活性位点(Gln, Met, Phe)-油腻或不油腻-始终比其他取代（His, Ser, Ala, Lys）发现的充满水的氧阴离子孔表现更好。虽然氧阴离子稳定似乎对HG3.17的效率有一定的贡献，但Gln50的作用在机制上比最初想象的要复杂得多，这强调了未来设计酶促氧阴离子孔的多因素方法的重要性。
Important reactions in biology and biocatalysis involve proton abstraction from s carbon. When the resulting anionic charge is delocalized from carbon to an oxygen atom, these deprotonations can be catalytically accelerated by oxyanion stabilization. Oxyanion stabilization by a glutamine side chain (Gln50) was thought to accelerate C-H proton abstraction in HG3.17, a computationally designed biocatalyst that had been evolutionarily optimized to enzyme-like efficiency. We present kinetic data and crystal structures at atomic resolution for six Gln50 mutants that indicate a surprisingly small advantage of the hydrogen-bond donor glutamine over "greasy" methionine. However, tightly packed active sites (Gln, Met, Phe)-greasy or not-perform consistently better than water-filled oxyanion holes found with other substitutions (His, Ser, Ala, Lys). Although oxyanion stabilization appears to contribute modestly to HG3.17 efficiency, the role of Gln50 is mechanistically more complex than initially thought, underscoring the importance of multifactorial approaches for the design of enzymatic oxyanion holes in the future.