Drug metabolism may be classified into two major pathways, Phase I and Phase II. The phase I pathways are comprised of oxidoreductase reactions, typically catalyzed by cytochrome P450, which can lead to substrates for subsequent phase II metabolism. The glutathione S- transferases (GSTs) are one of the several detoxification enzymes of phase II metabolic pathways. These enzymes catalyze the nucleophilic conjunction of co-factor glutathione to various electrophiles and xenobiotics. The resulting conjugates are usually less toxic and water- soluble compounds that can be excreted from the body. Because of their implication in cytotoxic drug resistance, GSTs are an important target of chemotherapy. Specific inhibitors of GSTs can be envisioned as potential candidates for chemotherapy adjuvants. The long term-goal of the research proposal is to understand the molecular mechanisms of GSTs in sufficient detail to allow rational design of inhibitors that are isozyme-specific.
The specific aims of this research proposal are (1) to optimize the expression of human GST A1-1 and GST P1-1 via E. coli expression vector systems, (2) to determine the chemical and kinetic competence of A- and P-class GSTs in hydrolyzing GSH thiol esters, and (3) to search for protein adduct corresponding to acylated P-class GSTs in hydrolyzing GSH thiol esters, and (3) to search for protein adduct corresponding to acylated GST. The various techniques in microbiology, molecular biology, enzyme kinetics, chromatography and mass spectrometry will be utilized to achieve each of these specific aims, leading to a better understanding of the catalytic and chemical mechanism of thiol ester hydrolysis by GST.
| Ibarra, Catherine A; Chowdhury, Pramit; Petrich, Jacob W et al. (2003) The anomalous pKa of Tyr-9 in glutathione S-transferase A1-1 catalyzes product release. J Biol Chem 278:19257-65 |
| Ibarra, Catherine; Grillo, Mark P; Lo Bello, Mario et al. (2003) Exploration of in vitro pro-drug activation and futile cycling by glutathione S-transferases: thiol ester hydrolysis and inhibitor maturation. Arch Biochem Biophys 414:303-11 |
| Nieslanik, B S; Ibarra, C; Atkins, W M (2001) The C-terminus of glutathione S-transferase A1-1 is required for entropically-driven ligand binding. Biochemistry 40:3536-43 |
| Ibarra, C; Nieslanik, B S; Atkins, W M (2001) Contribution of aromatic-aromatic interactions to the anomalous pK(a) of tyrosine-9 and the C-terminal dynamics of glutathione S-transferase A1-1. Biochemistry 40:10614-24 |
