Date of Award
8-2019
Document Type
Thesis
Degree Name
Master of Science (MS)
College/School
College of Science and Mathematics
Department/Program
Chemistry and Biochemistry
Thesis Sponsor/Dissertation Chair/Project Chair
Nina Goodey
Committee Member
John Siekierka
Committee Member
David Rotella
Committee Member
Saliya Desilva
Abstract
In drug discovery, building a comprehensive picture of the binding events between a drug and its enzyme target can be useful, especially in the development of new drugs or predicting the effect of a drug on a similar target from the same protein family. It is well known that a drug's selectivity is influenced by its interactions with amino acid residues within the active site, but contributions from residues situated away from (distal to) the active site are less well understood. Using a novel predictive approach PAnPredictor that employs sequence alignments, we have previously predicted residue positions in the dihydrofolate reductase (DHFR) family that play a role in drug selectivity. Interestingly, while a few of these predicted residues are located within the active site and are known drug specificity determining positions, other residues fall spatially in clusters distal to the active site. We used Bacillus stearothermophilus dihydrofolate reductase (Bs DHFR) to study the effect that introducing amino acid replacements at these predicted clusters has on the drug selectivity profile. Mutations were introduced randomly into each individual cluster and screened for functional DHFR enzymes. We determined kcat and KM values for the mutant enzymes that passed the functional screen to confirm that these mutants are functioning properly. Next, we determined KD values of the mutant enzymes to two common DHFR competitive drugs trimethoprim (TMP), and pyrimethamine (PYR) using fluorescence equilibrium titrations to identify residues where perturbations resulted in altered drug selectivity profiles. These data will help us understand the effect that distal residues have on drug selectivity and inform us on the ability of the predictive tool, PAnPredictor, to successfully identify Bacillus stearothennophilus dihydrofolate reductase determining residue positions in the DHFR enzyme family. If we find the approach successful, we plan to employ PAnPredictor to other protein families in the future.
File Format
Recommended Citation
Eck, Tyler, "The Effects of Active Site and Allosteric Residues on Catalysis and Inhibitor Selectivity in the Bacillus stearothennophilus Dihydrofolate Reductase" (2019). Theses, Dissertations and Culminating Projects. 323.
https://digitalcommons.montclair.edu/etd/323