Protein Science, Vol 6, Issue 4 873-881, Copyright © 1997 by Cold Spring Harbor Laboratory Press

ARTICLE

Pressure-dependent ionization of Tyr 9 in glutathione S-transferase A1-1: Contribution of the C-terminal helix to a ``soft'' active site

W. M. ATKINS, E. C. DIETZE and C. IBARRA
Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610

The glutathione S-transferase (GST) isozyme A1-1 contains at its active site a catalytic tyrosine, Tyr 9, which hydrogen bonds to, and stabilizes, the thiolate form of glutathione, GS(-). In the substrate-free GST A1-1, the Tyr 9 has an unusually low pK(a), ~8.2, for which the ionization to tyrosinate is monitored conveniently by UV and fluorescence spectroscopy in the tryptophan-free mutant, W21F. In addition, a short {alpha}-helix, residues 208-222, provides part of the GSH and hydrophobic ligand binding sites, and the helix becomes ``disordered'' in the absence of ligands. Here, hydrostatic pressure has been used to probe the conformational dynamics of the C-terminal helix, which are apparently linked to Tyr 9 ionization. The extent of ionization of Tyr 9 at pH 7.6 is increased dramatically at low pressures (p(1/2) = 0.52 kbar), based on fluorescence titration of Tyr 9. The mutant protein W21F:Y9F exhibits no changes in tyrosine fluorescence up to 1.2 kbar; pressure specifically ionizes Tyr 9. The volume change, {Delta}V, for the pressure-dependent ionization of Tyr 9 at pH 7.6, 19{deg}C, was -33 +/- 3 mL/mol. In contrast, N-acetyl tyrosine exhibits a {Delta}V for deprotonation of -11 +/- 1 mL/mol, beginning from the same extent of initial ionization, pH 9.5. The pressure-dependent ionization is completely reversible for both Tyr 9 and N-acetyl tyrosine. Addition of S-methyl GSH converted the ``soft'' active site to a noncompressible site that exhibited negligible pressure-dependent ionization of Tyr 9 below 0.8 kbar. In addition, Phe 220 forms part of an ``aromatic cluster'' with Tyr 9 and Phe 10, and interactions among these residues were hypothesized to control the order of the C-terminal helix. The amino acid substitutions F220Y, F220I, and F220L afford proteins that undergo pressure-dependent ionization of Tyr 9 with {Delta}V values of 31 +/- 2 mL/mol, 43 +/- 3 mL/mol, and 29 +/- 2 mL/mol, respectively. The p(1/2) values for Tyr 9 ionization were 0.61 kbar, 0.41 kbar, and 0.46 kbar for F220Y, F220I, and F220L, respectively. Together, the results suggest that the C-terminal helix is conformationally heterogeneous in the absence of ligands. The conformations differ little in free energy, but they are significantly different in volume, and mutations at Phe 220 control the conformational distribution.
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