Insight into mechanisms of allosteric regulation and cooperativity from yeast chorismate mutase
Open Access
- Author:
- Gorman, Scott
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 12, 2019
- Committee Members:
- David D Boehr, Dissertation Advisor/Co-Advisor
Philip C Bevilacqua, Committee Member
Scott A Showalter, Committee Member
David D Boehr, Committee Chair/Co-Chair
Amie Kathleen Boal, Outside Member
Philip C Bevilacqua, Program Head/Chair - Keywords:
- Enzyme
Allostery
Dynamics
NMR
ITC
Isothermal Titration Calorimetry
Nuclear Magnetic Resonance
Protein
Biophysics
Chorismate Mutase
ScCM - Abstract:
- Metabolism is the sum total of all of the biochemical reactions that take place in a living organism. These reactions include those involved in the breakdown of food to provide energy as well as the biosynthesis of biomolecules. Given the limited resources available within the cellular environment, biology has evolved various methods of regulating these biosynthetic processes. One important method of regulating biosynthetic pathways is allosteric regulation, in which the proteins involved in key pathway steps are able to alter their activities in response to varying levels of downstream metabolites. Saccharomyces cerevisiae chorismate mutase (ScCM) is an allosteric protein that catalyzes the isomerization of chorismate to prephenate via a Claisen rearrangement. This biochemical reaction represents the first committed step in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine, and is in competition with anthranilate synthase, which converts chorismate to anthranilate in the first committed step of tryptophan biosynthesis. Due in part to the high cost of tryptophan biosynthesis, regulation of ScCM by the pathway end products tyrosine and tryptophan is particularly important in yeast metabolism. Tyrosine and tryptophan allosterically inhibit and activate ScCM, respectively. Under some circumstances, there is also positive cooperativity for the substrate chorismate. As such, ScCM accounts for most of the different types of allosteric mechanisms, indicating that it is an excellent model system for understanding allosteric regulation. The studies contained within this thesis were performed with the goal of enhancing our understanding of the role and mechanisms of allosteric regulation of biosynthetic enzymes using ScCM as a model system. Towards this end, nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry were the most heavily used techniques. One important finding was that ScCM displays negative cooperativity between its effector binding sites, which results in its ability to adopt six distinct states in a cellular environment. This negative cooperativity gives ScCM the ability to have its activity fine-tuned in response to fluctuating levels of tyrosine and tryptophan. Another finding suggests the importance of water in facilitating negative cooperativity in the effector binding region. As ScCM binds a second effector ligand, many water molecules become trapped, resulting in a large entropic penalty. Finally, preliminary NMR studies on the ps-ns and µs-ms timescale dynamics of ScCM suggest that active site dynamics on the fast timescale are altered more by tyrosine than tryptophan, while dynamics on the slow timescale are altered more by tryptophan than tyrosine. These results indicate that modulation of entropy is important in determining allosteric inhibition, while modulation of structural dynamics is more important for allosteric activation. Overall this thesis makes the case for ScCM as a model system for understanding allostery, laying the ground work for understanding the roles of solvent entropy and enzyme structural dynamics in allosteric regulation.