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A Highly Efficient Artificial Hydrolase with a Well-Defined Dynamic Active Center
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AI
DOI：10.1021/acsami.6c03903.png)
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Abstract
En 中文
Although AI-based computational design can now produce de novo hydrolases with a well-defined structure, current de novo designed hydrolases and hydrolase mimics from all other approaches only exhibit low catalytic turnover numbers (kcat) that are several orders of magnitude lower than natural hydrolases, indicating that some essential features of natural enzymes are missing in current enzyme-mimicking and design. One important feature of natural enzymes that has never been captured in enzyme-mimicking and design is the fast dynamics of enzymes’ active centers, which is also the most challenging feature to mimic. Another important feature largely missed is the synergy among the catalytic triad, the oxyanion hole, and the binding site of enzymes. Herein, we create a gold nanoparticle (AuNP)-based artificial hydrolase, Goldenzyme, using a special conformational engineering (CE) approach to reconstruct the catalytic triad, the oxyanion hole, and the substrate binding site of α-chymotrypsin (α-CT) on AuNPs. NMR experiments demonstrate that Goldenzyme possesses a fast-dynamic feature, about 5-fold faster than a typical α-helix. Mutation experiments unambiguously demonstrate the essential roles of each key residue and the synergy among the catalytic triad, the oxyanion hole, and the binding site of Goldenzyme. Consequently, Goldenzyme efficiently hydrolyzes p-nitrophenyl acetate with a net kcat of 6.4 s–1 per active center, which is larger than that of α-CT (1.2 s–1). Remarkably, Goldenzyme can even hydrolyze some phthalate esters (PAEs)─tough nonactivated esters that cause serious environmental problems─while α-CT cannot. These results highlight the potential of CE, which has been successfully extended to silica NPs.
Keywords:
Catalytic activity
Hydrolysis
Organic compounds
Peptides and proteins
artificial enzyme
catalytic triad
conformational engineering
dynamic conformation
nanoparticle
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