On the origins of catalysis by ketosteroid isomerase
Abstract/Contents
- Abstract
- Enzymes are complicated molecular machines that actuate nearly all biochemical processes with speed and specificity -- their failure to do so nearly always has pathological consequences. Nevertheless, the origins of enzymes' prodigious catalytic power remain elusive and hotly debated despite the abundance of structural data and intense research efforts spanning the previous 60 years. This doctoral dissertation pertains largely to studies on the small enzyme, ketosteroid isomerase (KSI). KSI is among the most well studied enzymes, and has been used by many as a model for interrogating the catalytic strategies in Nature's toolbox. Its privileged status is warranted on account of it being one of the fastest enzymes known, accelerating its reaction's rate by approximately one trillion times, and doing so using a commonly encountered mechanism in biochemistry. In the ensuing chapters, we shall delve into the active site of KSI with a broad range of tools ranging from traditional biochemistry and spectroscopy, to analytical theory and computer simulation. Two complementary approaches are presented that break down KSI's catalysis into physical terms: in one approach, a thermodynamic framework is constructed that connects empirical macroscopic information (such as binding constants and pKa's) to the rate enhancement in a rigorous manner, inspired by and extending the classic model developed by Linus Pauling in 1946. In a second approach, the electric fields KSI's active site exerts onto its bound substrate are measured using vibrational Stark effect spectroscopy. From those measurements, I show that electric fields quantitatively explain the physical basis for a substantial fraction of KSI's catalytic power. The remaining catalytic effect is readily explained from separate measurements, as discussed. This analysis provides a decomposition of KSI's catalytic effect into microscopic catalytic strategies. Both approaches (thermodynamic and microscopic), while developed on KSI as a model, should be generally applicable to a wide range of enzymes. Electrostatic interactions have been broadly purported to play an essential role in catalysis; however, this dissertation presents the first direct experimental evidence that tests this proposal in a quantitative fashion. To measure electric fields inside proteins, I have taken advantage of the fact that the resonant frequencies of certain molecular vibrations are modulated in a predictable fashion by the local electric field experienced by the vibration (the vibrational Stark effect, VSE). A considerable number of methodological and conceptual developments of the VSE approach were needed to apply it toward interrogating the active site of KSI; these tools were first benchmarked on several simpler systems. In summary, this dissertation reports a reductionist and possibly comprehensive picture of KSI's catalysis. I also draw connections between electric field catalysis and several other prevailing theories about enzyme catalysis, and in particular demonstrate that it is intimately related to the concept of preorganization. I also propose general principles behind enzyme catalysis, which can provide insights, inter alia, for the design of artificial enzymes. The latter may prove some day to have many applications in medicine, energy, and the environment.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2014 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Fried, Stephen D |
|---|---|
| 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 | Fayer, Michael D |
| Thesis advisor | Solomon, Edward I |
| Advisor | Fayer, Michael D |
| Advisor | Solomon, Edward I |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Stephen D. Fried. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2014. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2014 by Stephen David Fried
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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