Dissecting proton delocalization and the electrostatic contribution to catalysis in ketosteroid isomerase with non-canonical amino acids
Abstract/Contents
- Abstract
- The origin of enzymes' catalytic power is a long-standing and still largely unsolved problem in biochemistry. To compare and evaluate the role of various catalytic strategies, I have turned to a model protein ketosteroid isomerase (KSI). Low barrier hydrogen bonds (LBHB) and electrostatic stabilization are two popular theories proposed to explain KSI's catalytic proficiency. The primary difference between the two hypotheses lies in the nature of the interaction between the transition state (TS) of the reaction and the extended H-bond network in KSI's active site, which preferentially stabilizes the TS to contribute to catalysis − in a LBHB formulation, the TS would enjoy an additional energetic benefit from the presumed covalent (delocalized) nature of a strong H-bond. To enable a side-by-side comparison of the two hypotheses, I utilized amber suppression and prepared a set of 'conservative mutants' by incorporating noncanonical amino acid 3-chlorotyrosine to each of the three tyrosine residues (Y16, Y57, Y32, denoted as 'tyrosine triad') within the extended H-bond network of KSI. The site-specific incorporation enabled the incremental and simultaneous tuning of the proton affinities and the electrostatic properties of KSI's active site, as suggested by small but systematic changes in KSI's catalytic rate. Moreover, X-ray crystal structures of the mutants verified the preservation of the extended H-bond network with minimal structural perturbation. The series of conservative mutants allows me to (1) dissect the quantum delocalization of protons within the extended H-bond network of apo-KSI; (2) critically test the linear correlation between KSI's electric field and its catalytic proficiency, reinforcing the dominant contribution of electrostatics in KSI catalysis; (3) and elucidate the functional connection between the strength of the active site H-bond and the electric field it exerts, suggesting a potential unification of the two hypotheses on the origin of enzymes' catalytic power.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2017 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Wu, Yufan |
|---|---|
| Associated with | Stanford University, Department of Chemistry. |
| Primary advisor | Boxer, Steven G. (Steven George), 1947- |
| Thesis advisor | Boxer, Steven G. (Steven George), 1947- |
| Thesis advisor | Cegelski, Lynette |
| Thesis advisor | Markland, Thomas E |
| Advisor | Cegelski, Lynette |
| Advisor | Markland, Thomas E |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Yufan Wu. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2017. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2017 by Yufan Wu
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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