Graduation Semester and Year
2021
Language
English
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Chemistry
Department
Chemistry and Biochemistry
First Advisor
Jongyun Heo
Abstract
Ras guanine nucleotide exchange factors (RasGEFs), which include Son of Sevenless (SOS), Ras Guanine Nucleotide Releasing Protein (RasGRP), and Ras Guanine Nucleotide Releasing Factor (RasGRF), catalyze nucleotide exchange of Ras upon activating signals. Crystal structure analyses suggest that catalytic cavities of RasGEFs are not deep enough to uphold their substrates. Given that RasGEFs are all membrane binding proteins, a novel “membrane supplement hypothesis” is proposed that RasGEFs in cytoplasm are catalytically unformed; and RasGEFs on membrane are catalytically competent. The reason RasGEFs are catalytically unformed without the support of membrane is because the catalytic sites of RasGEFs are not sufficiently deep to hold their substrates within their catalytic sites for their catalysis. It was shown that the catalytic function of SOS, one of RasGEFs, is enhanced upon its binding to membrane, supporting this membrane supplement hypothesis. To examine the membrane supplement hypothesis, an engineered RasGRP is designed, in which a catalytically unformed RasGRP is fused with a foreign protein that mimics the plasma membrane. Comparative kinetic analyses of the catalytically unformed RasGRP with the engineered RasGRP showed that the fused-foreign protein of engineered RasGRP functioned to hold and precisely orientate the substrate Ras within the catalytic site of engineered RasGRP, thereby making the engineered RasGRP become catalytically efficient. RasGEFs are classified as amphitropic enzymes, which adapt a compact conformation in the cytosol and are fully activated when recruited to membrane. The catalytic sites of many amphitropic proteins, including RasGEFs, are brought proximally toward the membrane interface after membrane binding event, and furthermore, the catalysis of these amphitropic enzymes is enhanced upon their membrane binding. However, the role of the membrane in the kinetic action of these amphitropic enzymes has not been previously proposed. The membrane supplement hypothesis in this study provides a rationalization that these amphitropic enzymes also possess a shallow pocket at their catalytic sites, challenging them to hold the bulky substrates. Therefore, locating the catalytic site of these amphitropic enzymes close to the membrane interface strengthens their substrate binding interactions, thereby facilitating their catalytic functions.
Keywords
Ras, RasGEFs, RasGRP, SOS, Membrane mimic, Engineered RasGRP, Catalytically unformed RasGEFs, Catalytically competent RasGEFs, Amphitropic proteins, Amphitropic enzymes, Disulfide bridge, Nucleotide exchange, Kinetic constants
Disciplines
Chemistry | Physical Sciences and Mathematics
License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Hoang, Hanh My, "DETERMINATION OF THE KINETIC ROLES OF THE MEMBRANE IN THE CATALYSIS OF RASGEFS BY USING AN ENGINEERED MODEL PROTEIN" (2021). Chemistry & Biochemistry Dissertations. 201.
https://mavmatrix.uta.edu/chemistry_dissertations/201
Comments
Degree granted by The University of Texas at Arlington