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