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Examining how allosteric mutations affect ligand binding and specificity on Dihydrofolate reductase
Author(s) -
Alfonso Melany,
Okondo Marian,
Goodey Nina M
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.760.10
Subject(s) - dihydrofolate reductase , allosteric regulation , biochemistry , mutant , enzyme , alanine , biology , antifolate , active site , chemistry , amino acid , methotrexate , gene , antimetabolite , immunology
The purpose of my research is to study the enzyme Dihydrofolate reductase (DHFR) from the organism Bacillus stearothermophillus (Bs). DHFR is an important enzyme because it catalyzes the reduction of 7,8‐dihydrofolate (DHF) to 5,6,7,8‐tetrahydrofolate (THF) and THF is essential for DNA synthesis. For this reason, DHFR is the subject of many studies and is a pharmaceutical drug target to treat diseases like cancer and third world bacterial and protozoal infections. Drugs like methotrexate (MTX), pyrimathemine (PYR), and trimethoprim (TMP) are used to inhibit DHFR homologs from different pathogens and treat diseases caused by these pathogens. In many cases, however, it has been found that DHFR builds resistance to these antifolate drug compounds. Previously, it was found by Dr. Goodey and her students that drug binding and selectivity in DHFR has to do with the amino acid residue mutations in both the active site and in allosteric regions on the DHFR. For this project, we want to understand how these allosteric mutations affect ligand binding and specificity. The research was started by making two mutants (each have one allosteric mutation) and comparing them to the DHFR wild type. The first mutation distal from the active site was isoleucine in position 86 to alanine (I86A) and tyrosine in position 127 to alanine (Y127A). These DHFR mutants have been purified and a fluorescent dye molecule that act as a reporter tag was added to them. From these fluorescently labeled DHFR proteins I have worked on understanding the effect of these mutations on conformational motions associated with drug binding and selectivity by measuring the turnover number ( k cat ) and dissociation constant (K d ) of these proteins.