Thomas McHugh

Project title: Metabolism of Aflatoxin Epoxide by Glutathione S-transferase: New Insights into GST Function

Degree: PhD | Program: Environmental Toxicology (Tox) | Project type: Thesis/Dissertation
Completed in: 1997 | Faculty advisor: David L. Eaton


Glutathione S-Transferases (GSTs) are a family of broad substrate specificity detoxification enzymes which specialize in the metabolism of reactive substrates with electrophilic functional groups. Even closely related GST isoenzymes can have large differences in activity towards specific substrates. For examples, mGSTA3-3 and rGSTA3-3 have activity towards aflatoxin-8,9-exo-epoxide (AFBO) of 265 and <0.2 nmol/mg/min despite being 86% identical and having similar activity towards the model substrate 1-chloro-2,4-dinitrobenzene. Site directed mutagenesis has been used to determine the structural features in GST which are required for metabolism of AFBO. rGSTA3-3 isoenzymes with one or two mutations have little or no activity towards AFBO, however a six site mutant activity of 40 nmol/mg/min, a greater than 200 fold increase over the wild-type enzyme. In contrast to previous studies of activity differences in GST isoenzymes which identified two or three critical amino acid resides (Shan and Armstrong 1994; Bammler et al. 1995), at least seven non-conserved amino acids residues contribute to high activity towards AFBO in mGSTA3-3.

GSTs are dimeric enzymes composed of two identical or closely related subunits. Previous studies have found that the two subunits act independently in the metabolism of small model substrates (Danielson and Mannervik 1985). To date, no functional advantage has been identified for GST dimerization. The binding of two different reaction products (p-nitrobenzyl-glutathione, the aflatoxin-glutathione, AFB-GHS) to mGSTA3-3 has been measured using equilibrium dialysis. As expected, p-nitrobenzyl-glutathione was found to bind with a stoichiometry of 2.24+0.17 mole per mole of dimeric enzyme. However, the much larger aflatoxin-glutathione conjugate (AFB-GSH), was found to bind with a stoichiometry of 1.12+0.08 mole per mole of dimeric enzyme. This is the first report of a GST reaction product which binds to the enzyme with a stoichiometry of 1 mole per mole of dimmer. As a follow-up, the novel heterodimer, mGSTA3-rGSTA3, was expressed, purified, and characterized. The heterodimer was functional and exhibited activities towards ethacrynic acid, 1-chloro-2,4-dinitrobenzene, and aflatoxin-exo-8,9-epoxide (AFBO) which were 101%, 66% and 61% of those predicted from the homodimers, respectively. In the case of AFBO, the decrease in activity was associated with an equivalent decrease in the rate of product release from the enzyme. These results demonstrate that dimerization affects activity of GST subunits.