Kinetic Studies of Glutathione S-Transferase
Date of Award
Master of Science (MS)
Benjamin J. Alper, Ph.D.
Glutathione S-Transferase (GSTs) are important metabolic enzymes with roles in drug detoxification and elimination in humans and other organisms. Here we present the enzymatic characterization of bacterially-derived recombinant GST from the human parasite Schistosoma japonicum. Kinetic parameters of S. japonicum GST function were determined using an established colorimetric assay that was here adapted for high-throughput study. Inhibition of S. japanicum GST catalytic activity was shown using p-aminobenzoic acid, a recognized GST inhibitor. The colorimetric assay was subsequently applied to demonstrate inhibition of S. japonicum GST by α-fluoromethylhistidine, a novel GST inhibitor that was identified using in silico screening methods. On the basis of kinetic studies including inhibitor profiling experiments, the bisubstrate mechanism of S. japonicum GST was established. S. japonicum GST was found to exhibit a random sequential substrate binding mechanism, consistent with the behavior of related μGST isoforms. Last, using the high-throughput GST colorimetric assay, a laboratory experiment was developed for the undergraduate biochemistry laboratory. This experiment introduced techniques and models for characterization of kinetic mechanisms of bisubstrate enzymes, which are among the most abundant enzymes found in nature.
Stefanidis, Lazaros, "Kinetic Studies of Glutathione S-Transferase" (2017). Chemistry Master’s Theses. 41.