The functional role of Phe-10 and the anomalous Tyr-9 pK(a) in glutathione s-transferase A1-1

As the most abundant isoform of GST in the liver, the GST A1-1 is the major catalyst of detoxification. This isoform is of particular interest because it exhibits an unusually low pK_a (∼8.1–8.3) of active site Tyr-9 and a ligand-dependent dynamics of the C-terminus. This dynamics is catalytically important, as product release is rate-limiting for a number of substrates.; A major emphasis of this dissertation is the elucidation of the structural and functional significance of the anomalous pK_a of Tyr-9 in rat GST A1-1. The edge-to-face electrostatic interaction between Phe-10 and Tyr-9 contributes to the ionization properties of the tyrosyl hydroxyl group in the apo-enzyme, consistent with crystal structure observations. Additionally, Phe-10 plays a vital role in ligand binding, as the two steps of the binding reaction are significantly reduced upon removal of aromatic-aromatic interactions. The results presented herein demonstrate the importance of Phe-10 in orchestrating the dynamics of the C-terminus.; Previous data suggest that Tyr-9 ionization is closely coupled to the C-terminal dynamics of GST Al-1. To directly probe this possibility, engineered W21F:F222W and Y9F:W21F:F222W mutants, which contain single Trp at the C-terminus, were used as fluorescent reporters of C-terminal dynamics. Only the W21F:F222W mutant exhibited pH-dependent change in Trp-222 emission properties consistent with changes in C-terminal solvation. Moreover, the pH-dependent equilibrium binding and stopped-flow studies with product conjugate resulted in decreased equilibrium binding affinity (K_D), concomitant with increased binding and dissociation rates at higher pHs. The recovered pK_as for the change in microscopic rate constants reflect the pK_a of Tyr-9 in the apo-enzyme. In contrast, these pH-dependent changes were not observed in the Y9F:W21F:F222W mutant. These results demonstrate that the ionization status of Tyr-9 controls the C-terminal dynamics, providing parallel pathways for product dissociation.; Finally, the utility of GST-mediated hydrolysis of thiol esters formed from glutathione (GSH) and several carboxylic acid-containing drugs were exploited as a means of delivering drugs to tumors that overexpress GSTs. The competence of several human GSTs was examined, with the Pi-class GST being the most active in hydrolyzing the thiol ester formed from ethacrynic acid (EA) and GSH. Moreover, the P-class GST was inhibited and modified by the released EA. Results also indicate the importance of active site Tyr in the Alpha-, Pi-, and Mu-class GSTs. However, the exact mechanism by which this residue participates in the hydrolysis remains unknown.