酯水解酶中多个活性位点的合理工程,Biochemistry

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酯水解酶中多个活性位点的合理工程
Biochemistry ( IF 2.9 ) Pub Date : 2018-03-30 00:00:00 , DOI: 10.1021/acs.biochem.8b00274
Gerard Santiago ₁ , Mónica Martínez-Martínez ₂ , Sandra Alonso ₂ , Rafael Bargiela ₂ , Cristina Coscolín ₂ , Peter N. Golyshin , Víctor Guallar _{1,

3} , Manuel Ferrer ₂

Affiliation

广泛报道了改变酶促均相催化剂中活性位点性质的影响。然而，仍未探索增加这种位点数目的可能性，如在非均相催化材料中通常所做的那样，特别是因为那些位点必须以特定构型容纳适当的残基。通过使用丝氨酸酯水解酶作为靶酶研究了这种可能性。通过使用可映射配体扩散和结合的蛋白质能量景观探索软件，我们发现了一个潜在的结合口袋，能够容纳额外的催化三元组和氧阴离子空穴接触。通过引入两个突变，该结合口袋成为催化位点。其底物特异性，底物偏爱，由于活性位点结构的不同，其催化活性与野生型酯水解酶和其他水解酶的天然位点不同。已证明将结合袋转变成额外的催化活性位点是创建具有两个功能性反应基团的丝氨酸酯水解酶的成功方法。我们的结果说明了现代建模技术的准确性和可预测性，为单一酶中存在不同催化环境带来了新的催化机会。

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Rational Engineering of Multiple Active Sites in an Ester Hydrolase

Effects of altering the properties of an active site in an enzymatic homogeneous catalyst have been extensively reported. However, the possibility of increasing the number of such sites, as commonly done in heterogeneous catalytic materials, remains unexplored, particularly because those have to accommodate appropriate residues in specific configurations. This possibility was investigated by using a serine ester hydrolase as the target enzyme. By using the Protein Energy Landscape Exploration software, which maps ligand diffusion and binding, we found a potential binding pocket capable of holding an extra catalytic triad and oxyanion hole contacts. By introducing two mutations, this binding pocket became a catalytic site. Its substrate specificity, substrate preference, and catalytic activity were different from those of the native site of the wild type ester hydrolase and other hydrolases, due to the differences in the active site architecture. Converting the binding pocket into an extra catalytic active site was proven to be a successful approach to create a serine ester hydrolase with two functional reactive groups. Our results illustrate the accuracy and predictive nature of modern modeling techniques, opening novel catalytic opportunities coming from the presence of different catalytic environments in single enzymes.

更新日期：2018-03-30

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