MUTATION OF AN EVOLUTIONARILY CONSERVED TYROSINE RESIDUE IN THE ACTIVE-SITE OF A HUMAN CLASS ALPHA-GLUTATHIONE TRANSFERASE

Human class Alpha glutathione transferase (GST) A1-1 has been subjected to site-directed mutagenesis of a Tyr residue conserved in all classes of cytosolic GSTs. The change of Tyr8 --> Phe lowers the specific activities with three substrates to 2-8% of the values for the wild-type enzyme. The changes in the kinetic parameters k(cat)/K(M), V(max) and S0.5 show that the decreased activities are partly due to a reduced affinity for glutathione. The effect is reflected in lowered k(cat) values, suggesting that the hydroxyl group of Tyr8 is involved in the activation of glutathione. The proposal of such a role for the Tyr residue has support from the 3D structure of a pig lung class Pi GST [Reinemer et al. (1991) EMBO J. 10, 1997-2005]. Thus, Tyr8 appears to be the first active site residue established as participating in the chemical mechanism of a GST.