ArticleProceedings of the National Academy of Sciences of the United States of America2011
Design of a switchable eliminase.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 54 papers.
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Who cites it
54 citing papers in PubMed.
- Emergence of specific binding and catalysis from a designed generalist binding protein.Nature chemistry · 2026Article
- How far can you go? Extrapolating values of catalytic activity from known protein landscapes in natural and directed evolution.Chemical Society reviews · 2026Review
- Enzyme Enhancement Through Computational Stability Design Targeting NMR-Determined Catalytic Hotspots.Journal of the American Chemical Society · 2025Article
- Fishing for Catalysis: Experimental Approaches to Narrowing Search Space in Directed Evolution of Enzymes.JACS Au · 2023Review
- Computation-Aided Engineering of Cytochrome P450 for the Production of Pravastatin.ACS catalysis · 2022Article
- Convergence in determining enzyme functional descriptors across Kemp eliminase variants.Electronic structure (Bristol, England) · 2022Article
- NMR-guided directed evolution.Nature · 2022Article
- Active site center redesign increases protein stability preserving catalysis in thioredoxin.Protein science : a publication of the Protein Society · 2022Article
- Phosphorylation promotes the endonuclease-like activity of human centrin 2.RSC advances · 2022Article
- Rational design of allosteric switchable catalysts.Exploration (Beijing, China) · 2022Review
- How directed evolution reshapes the energy landscape in an enzyme to boost catalysis.Science (New York, N.Y.) · 2020Article
- Enhancing aChemical science · 2020Article
- MinimalistACS catalysis · 2019Article
- Article
- A Designed Enzyme Promotes Selective Post-translational Acylation.Chembiochem : a European journal of chemical biology · 2018Article
- Protein Environment: A Crucial Triggering Factor in Josephin Domain Aggregation: The Role of 2,2,2-Trifluoroethanol.International journal of molecular sciences · 2018Article
- Conformational dynamics and enzyme evolution.Journal of the Royal Society, Interface · 2018Review
- Role of conformational dynamics in the evolution of novel enzyme function.Chemical communications (Cambridge, England) · 2018Review
- Rational and Semirational Protein Design.Methods in molecular biology (Clifton, N.J.) · 2018Review
- De novo active sites for resurrected Precambrian enzymes.Nature communications · 2017Article
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Authors and funding
6 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The active sites of enzymes are lined with side chains whose dynamic, geometric, and chemical properties have been finely tuned relative to the corresponding residues in water. For example, the carboxylates of glutamate and aspartate are weakly basic in water but become strongly basic when dehydrated in enzymatic sites. The dehydration of the carboxylate, although intrinsically thermodynamically unfavorable, is achieved by harnessing the free energy of folding and substrate binding to reach the required basicity. Allosterically regulated enzymes additionally rely on the free energy of ligand binding to stabilize the protein in a catalytically competent state. We demonstrate the interplay of protein folding energetics and functional group tuning to convert calmodulin (CaM), a regulatory binding protein, into AlleyCat, an allosterically controlled eliminase. Upon binding Ca(II), native CaM opens a hydrophobic pocket on each of its domains. We computationally identified a mutant that (i) accommodates carboxylate as a general base within these pockets, (ii) interacts productively in the Michaelis complex with the substrate, and (iii) stabilizes the transition state for the reaction. Remarkably, a single mutation of an apolar residue at the bottom of an otherwise hydrophobic cavity confers catalytic activity on calmodulin. AlleyCat showed the expected pH-rate profile, and it was inactivated by mutation of its active site Glu to Gln. A variety of control mutants demonstrated the specificity of the design. The activity of this minimal 75-residue allosterically regulated catalyst is similar to that obtained using more elaborate computational approaches to redesign complex enzymes to catalyze the Kemp elimination reaction.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.