ArticleProceedings of the National Academy of Sciences of the United States of America2012
Engineering a model protein cavity to catalyze the Kemp elimination.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 25 papers.
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Who cites it
25 citing papers in PubMed.
- Emergence of specific binding and catalysis from a designed generalist binding protein.Nature chemistry · 2026Article
- 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
- NMR-guided directed evolution.Nature · 2022Article
- Enhancing aChemical science · 2020Article
- Enabling protein-hosted organocatalytic transformations.RSC advances · 2020Review
- MinimalistACS catalysis · 2019Article
- Article
- Role of conformational dynamics in the evolution of novel enzyme function.Chemical communications (Cambridge, England) · 2018Review
- De novo active sites for resurrected Precambrian enzymes.Nature communications · 2017Article
- A multi-systemic mitochondrial disorder due to a dominant p.Y955H disease variant in DNA polymerase gamma.Human molecular genetics · 2017Article
- A redox-mediated Kemp eliminase.Nature communications · 2017Article
- Kemp Eliminase Activity of Ketosteroid Isomerase.Biochemistry · 2017Article
- Hydrogen Bonding of 1,2-Azaborines in the Binding Cavity of T4 Lysozyme Mutants: Structures and Thermodynamics.Journal of the American Chemical Society · 2016Article
- Kemp Elimination in Cationic Micelles: Designed Enzyme-Like Rates Achieved through the Addition of Long-Chain Bases.Journal of physical organic chemistry · 2016Article
- Computational strategies for the design of new enzymatic functions.Archives of biochemistry and biophysics · 2015Review
- Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification.ACS chemical biology · 2014Article
- Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
- Catalytic efficiency of designed catalytic proteins.Current opinion in structural biology · 2014Review
- RB69 DNA polymerase structure, kinetics, and fidelity.Biochemistry · 2014Review
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2 authors.
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Abstract
Synthetic cavitands and protein cavities have been widely studied as models for ligand recognition. Here we investigate the Met102 → His substitution in the artificial L99A cavity in T4 lysozyme as a Kemp eliminase. The resulting enzyme had k(cat)/K(M) = 0.43 M(-1) s(-1) and a (k(cat)/K(M))/k(uncat) = 10(7) at pH 5.0. The crystal structure of this enzyme was determined at 1.30 Å, as were the structures of four complexes of substrate and product analogs. The absence of ordered waters or hydrogen bonding interactions, and the presence of a common catalytic base (His102) in an otherwise hydrophobic, buried cavity, facilitated detailed analysis of the reaction mechanism and its optimization. Subsequent substitutions increased eliminase activity by an additional four-fold. As activity-enhancing substitutions were engineered into the cavity, protein stability decreased, consistent with the stability-function trade-off hypothesis. This and related model cavities may provide templates for studying protein design principles in radically simplified environments.
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