ArticleBiochemistry2014
Experimental and computational mutagenesis to investigate the positioning of a general base within an enzyme active site.
Article in Biochemistry, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed, 21 citations in OpenAlex.
- Ensemble-function relationships to dissect mechanisms of enzyme catalysis.Science advances · 2022Article
- Accurate positioning of functional residues with robotics-inspired computational protein design.Proceedings of the National Academy of Sciences of the United States of America · 2022Article
- Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray-derived conformational ensembles.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
- Bottom-Up Nonempirical Approach To Reducing Search Space in Enzyme Design Guided by Catalytic Fields.Journal of chemical theory and computation · 2020Article
- Oscillatory Active-site Motions Correlate with Kinetic Isotope Effects in Formate Dehydrogenase.ACS catalysis · 2019Article
- Computational design of structured loops for new protein functions.Biological chemistry · 2019Review
- Electric Fields and Enzyme Catalysis.Annual review of biochemistry · 2017Review
- A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase.Journal of the American Chemical Society · 2016Article
- BIOPHYSICS. Response to Comments on "Extreme electric fields power catalysis in the active site of ketosteroid isomerase".Science (New York, N.Y.) · 2015Article
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Authors and funding
8 authors at 4 institutions in 1 country.
Funding
Abstract
The positioning of catalytic groups within proteins plays an important role in enzyme catalysis, and here we investigate the positioning of the general base in the enzyme ketosteroid isomerase (KSI). The oxygen atoms of Asp38, the general base in KSI, were previously shown to be involved in anion-aromatic interactions with two neighboring Phe residues. Here we ask whether those interactions are sufficient, within the overall protein architecture, to position Asp38 for catalysis or whether the side chains that pack against Asp38 and/or the residues of the structured loop that is capped by Asp38 are necessary to achieve optimal positioning for catalysis. To test positioning, we mutated each of the aforementioned residues, alone and in combinations, in a background with the native Asp general base and in a D38E mutant background, as Glu at position 38 was previously shown to be mispositioned for general base catalysis. These double-mutant cycles reveal positioning effects as large as 10(3)-fold, indicating that structural features in addition to the overall protein architecture and the Phe residues neighboring the carboxylate oxygen atoms play roles in positioning. X-ray crystallography and molecular dynamics simulations suggest that the functional effects arise from both restricting dynamic fluctuations and disfavoring potential mispositioned states. Whereas it may have been anticipated that multiple interactions would be necessary for optimal general base positioning, the energetic contributions from positioning and the nonadditive nature of these interactions are not revealed by structural inspection and require functional dissection. Recognizing the extent, type, and energetic interconnectivity of interactions that contribute to positioning catalytic groups has implications for enzyme evolution and may help reveal the nature and extent of interactions required to design enzymes that rival those found in biology.
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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.