U530034.pdf (12.52 MB)
Download file

Protein engineering to probe the catalytic mechanism of Alpha-class glutathione S-transferases

Download (12.52 MB)
posted on 15.12.2014, 10:32 by Paul David. McDonagh
Glutathione S-Transferases (GSTs) are a large family of isoenzymes that catalyse the addition of glutathione to hydrophobic electrophiles. Over-expression of GSTs in tumours leads to resistance to chemotherapy drugs, thus understanding GST biochemistry is clinically important. However, the catalytic and substrate recognition mechanisms remain poorly understood.;Two rat, alpha-class GSTs, Yc1 and Yc2 have 91% homology but have specific activities of 0.01 and 13.2 nmoles/min/mg respectively for the carcinogen aflatoxin-exo-8,9-epoxide. The protein structures for each were homology modelled on the co-ordinates for alpha-class human GSTA1-1. S-aflatoxinyl glutathione modelled into the active sites identifying positions 108 and 208 as important residues. A 'knock-out' double mutant D208MY108L was made in rGST Yc2 and an 'engineered' E208DH108Y mutant was made for Yc1.;The mutations reduced rGST Yc2 activity to <0.01 nmoles/min/mg and increased rGST Yc1 activity to 0.32 nmoles/min/mg which was further used to protect a human bronchial cell line against aflatoxin B1. Modelling of S-aflatoxinyl glutathione into huGSTA1-1 suggested the same positions were important in determining its low activity for aflatoxin-exo-8,9-epoxide. The double mutant L108YM208D failed to engineer any significant activity for aflatoxin-exo-8,9-epoxide into the enzyme.;The C-terminus of huGSTA1-1 was deleted and the kinetics of the truncated enzyme determined in the presence and absence of a synthetic peptide designed to replace the helix sequence. Kcat and Km were modified for the deleted enzyme in the presence of the peptide but Kcat/Km was not, suggesting the helix plays a part in promoting productive substrate binding.;The catalytically important Tyr9 was investigated by NMR. Tyr9 is thought be responsible, in part, for the deprotonation of glutathione during catalysis and as such must have a lower pKa than tyrosine in solution. Assignment of Tyr9 in the NMR spectrum allowed its titration, confirming that the pKa of Tyr9 is shifted from 10.0 to 7.720.21.


Date of award


Author affiliation


Awarding institution

University of Leicester

Qualification level


Qualification name




Usage metrics