Contribution of aromatic-aromatic interactions to the anomalous pK(a) of tyrosine-9 and the C-terminal dynamics of glutathione S-transferase A1-1.
Most cytosolic glutathione S-transferases (GSTs) exploit a hydrogen bond between an active site Tyr and the bound glutathione (GSH) cofactor to lower the pK(a) of the GSH and generate the nucleophilic thiolate anion, GS(-). In human (hGSTA1-1) and rat (rGSTA1-1) homologues, the active site Tyr-9 has a low pK(a) of 8.1-8.3, for which the functional significance is unknown. Crystal structures of GSTA1-1 suggest that weakly polar interactions between the electropositive ring edge of Phe-10 and the pi-cloud of Tyr-9, in the apoenzyme, could stabilize the tyrosinate anion and also modulate the pK(a) of GSH. Upon binding a product GSH conjugate, Phe-10 moves away from Tyr-9, allowing the highly dynamic C-terminus to "close" over the active site. To explore the role of Phe-10 in modulating the Tyr-9 pK(a) and in ligand binding, rGSTA1-1 mutants F10Y, F10L, and F10A were characterized. The pK(a)s of Tyr-9 in the apoenzymes were 8.2 +/- 0.2, 8.7 +/- 0.2, and 9.3 +/- 0.1, respectively, for F10Y, F10L, and F10A, compared to 8.3 +/- 0.2 for the "wild type". The experimentally determined pK(a)s qualitatively paralleled the energies required to remove a proton predicted by ab initio calculations using model compounds constrained to the coordinates of rGSTA1-1. The pK(a) of GSH in the binary complex was significantly less affected by these substitutions. In contrast, F220I and F220Y C-terminal mutations caused the pK(a) of Tyr-9 to decrease modestly. For the binary complex with S-hexyl-GSH, which induces the "closed" conformation, Tyr-9 retains a low pK(a) and the Phe-10 substitutions have significant effects. Presumably, Phe-10 plays a critical structural role in stabilizing the closed conformation. The mutations F10L and F10A also slowed the rate of GSH conjugate binding by 10-20-fold, as measured by stopped-flow fluorescence. The effects of Phe-10 substitution were large for both steps of the biphasic binding reaction, suggesting the importance of aromatic interactions throughout the reaction coordinate. A unified view of the C-terminal dynamics of GSTA1-1 is discussed, which emphasizes the coupling between Tyr-9 ionization, active site solvation, and C-terminal dynamics.
Ibarra
C
C
Department of Medicinal Chemistry, Box 357610, University of Washington, Seattle, Washington 98195-7610, USA.
Nieslanik
B S
BS
Atkins
W M
WM
D000818:Animals; D004926:Escherichia coli; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000378:metabolism*
D008024:Ligands; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D010649:Phenylalanine; D005982:Glutathione Transferase; D005978:Glutathione