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Effect of Non‐Active Site Amino Acid Residues on Inhibitor Selectivity
Author(s) -
Eck Tyler,
Gubler Ueli,
Goodey Nina
Publication year - 2018
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.2018.32.1_supplement.791.10
Subject(s) - active site , dihydrofolate reductase , binding site , amino acid , enzyme , biochemistry , chemistry , mutant , biology , stereochemistry , gene
Enzyme inhibitors are important in biological processes and as pharmaceutical drugs, the cornerstone of modern medicine. It is necessary to build a comprehensive picture of the binding event between the inhibitor and its enzyme target to effectively design pharmaceutical drugs and predict the binding of known drugs to new targets. It is currently known that inhibitor selectivity is in part determined by the amino acid residues within the “active site” , which is the site/cavity in the enzyme where the inhibitor binds. Less well understood is the role of residues situated away from (distal to) the active site and how they contribute to inhibitor selectivity, which refers to the fact that inhibitor molecules often bind to several members of an enzyme family but not all. By understanding the features that determine inhibitor binding within an enzyme family, it is possible to design better inhibitors and predict possible new targets for a given inhibitor. Using Bacillus stearothermophilus dihydrofolate reductase (Bs DHFR) as a model system, this project studies how amino acids distal from the active site affect inhibitor binding and selectivity. Previous studies in our lab have indeed predicted and experimentally verified three clusters of amino acids in Bs DHFR that are distal to the active site but impact inhibitor binding. Here, random mutations are being introduced individually into these clusters. Mutants will be screened for DHFR activity using a functional assay based on ability to synthesize thymidine. DHFR coding genes in selected colonies will be sequenced. Genes with functional amino acid mutations will be used to produce the corresponding DHFR enzyme variants. To ensure that the variants are active, k cat and K M values will be determined. To investigate the impact of the mutations on inhibitor selectivity, K D values will be determined for multiple inhibitors including Methotrexate (MTX), Trimethoprim (TMP), and Pyrimethamine (PMT). Analysis of these results will show how and how much distal residues affect Bs DHFR's inhibitor selectivity. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .