Experimental and Computational Mutagenesis to Investigate the Positioning of a General Base Within an Enzyme Active Site. Schwans, J. P, Hanoian, P., ˘nderlineLengerich, ˘., Sunden, F., Gonzalez, A., Tsai, Y., Hammes-Schiffer, S., & Herschlag, D. Biochemistry, 53(15):2541–2555, American Chemical Society, 2014. ![Experimental and Computational Mutagenesis to Investigate the Positioning of a General Base Within an Enzyme Active Site [link]](https://bibbase.org/img/filetypes/link.svg "link")
Paper abstract bibtex 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 103-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.
@article{schwans2014experimental,
title={Experimental and Computational Mutagenesis to Investigate the Positioning of a General Base Within an Enzyme Active Site},
author={Schwans, Jason P and Hanoian, Philip and \underline{Lengerich}, \underline{Benjamin J.} and Sunden, Fanny and Gonzalez, Ana and Tsai, Yingssu and Hammes-Schiffer, Sharon and Herschlag, Daniel},
journal={Biochemistry},
volume={53},
number={15},
pages={2541--2555},
year={2014},
informal_venue = {Biochemistry},
publisher={American Chemical Society},
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 103-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.},
url_paper = {https://pubs.acs.org/doi/pdf/10.1021/bi401671t},
keywords = {Molecular Dynamics, Computational Chemistry}
}
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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 103-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.","url_paper":"https://pubs.acs.org/doi/pdf/10.1021/bi401671t","keywords":"Molecular Dynamics, Computational Chemistry","bibtex":"@article{schwans2014experimental,\n title={Experimental and Computational Mutagenesis to Investigate the Positioning of a General Base Within an Enzyme Active Site},\n author={Schwans, Jason P and Hanoian, Philip and \\underline{Lengerich}, \\underline{Benjamin J.} and Sunden, Fanny and Gonzalez, Ana and Tsai, Yingssu and Hammes-Schiffer, Sharon and Herschlag, Daniel},\n journal={Biochemistry},\n volume={53},\n number={15},\n pages={2541--2555},\n year={2014},\n informal_venue = {Biochemistry},\n publisher={American Chemical Society},\n 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 103-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. 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