Molecular recognition of phosphatases : insights into specificity and the destabilizing influence of an enzyme active site
Abstract/Contents
- Abstract
- Chemical transfer of the phosphoryl group is perhaps the most critical and pervasive reaction in biology, and the enzymes that catalyze these reactions provide some of the largest reaction rate enhancements known. Phosphoryl transfer enzymes have evolved active sites with a remarkable ability to recognize and distinguish similar substrates and transient species along the reaction pathway. A full understanding of the physical features and recognition strategies used by these active sites is lacking and remains as a critical obstacle to the design of novel enzymes for applications in health and industry. All enzymes must provide preferential stabilization to reaction transition states while limiting binding to ground state substrates and products. Alkaline Phosphatase (AP) is one of Nature's most impressive examples, as it prefers stabilizing the phosphoryl group transition state by 10^22-fold compared to its modest binding of the phosphate monoester substrate in the ground state. Binding studies were carried out with a set of highly similar ground state molecules to probe this tremendous molecular discrimination. The results showed that despite the similarities of these molecules, AP has a very strong preference for binding inorganic phosphate (Pi). A vibrational spectroscopy approach was used to dissect the atomic details of Pi binding to AP. These measurements in addition to pH-dependent binding studies suggest that a proton from Pi is transferred to AP upon binding allowing it to bind more strongly than related molecules that lack a transferrable proton. It was proposed that the proton transfer neutralizes electrostatic repulsion between the anionic active site nucleophile of AP and the negatively charged oxygen atoms of the bound ligand. This model implicated a role for anionic active site nucleophiles, when left deprotonated, in destabilizing ground state binding and contributing to the enzyme's enormous preferential stabilization of the transition state. This previously unknown role for anionic nucleophiles was tested further using mutagenesis studies and binding affinity comparisons in AP and another phosphatase. The results provide new insights into how anionic nucleophiles of phosphoryl transfer enzymes contribute to catalysis.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2012 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Andrews, Logan Dane |
|---|---|
| Associated with | Stanford University, Department of Chemical and Systems Biology. |
| Primary advisor | Herschlag, Daniel |
| Thesis advisor | Herschlag, Daniel |
| Thesis advisor | Chen, James |
| Thesis advisor | Wandless, Thomas |
| Advisor | Chen, James |
| Advisor | Wandless, Thomas |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Logan Dane Andrews. |
|---|---|
| Note | Submitted to the Department of Chemical and Systems Biology. |
| Thesis | Ph.D. Stanford University 2012 |
| Location | electronic resource |
Access conditions
- Copyright
- © 2012 by Logan Dane Andrews
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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