title: The anomalous pKa of Tyr-9 in glutathione S-transferase A1-1 catalyzes product release.
abstract: The pKa of the catalytic Tyr-9 in glutathione S-transferase (GST) A1-1 is lowered from 10.3 to approximately 8.1 in the apoenzyme and approximately 9.0 with a GSH conjugate bound at the active site. However, a clear functional role for the unusual Tyr-9 pKa has not been elucidated. GSTA1-1 also includes a dynamic C terminus that undergoes a ligand-dependent disorder-to-order transition. Previous studies suggest a functional link between Tyr-9 ionization and C-terminal dynamics. Here we directly probe the role of Tyr-9 ionization in ligand binding and C-terminal conformation. An engineered mutant of rGSTA1-1, W21F/F222W, which contains a single Trp at the C terminus, was used as a fluorescent reporter of pH-dependent C-terminal dynamics. This mutant exhibited a pH-dependent change in Trp-222 emission properties consistent with changes in C-terminal solvation or conformation. The apparent pKa values for the conformational transition were 7.9 +/- 0.1 and 9.3 +/- 0.1 for the apoenzyme and ligand-bound enzyme, respectively, in excellent agreement with the pKa for Tyr-9 in these states. The Y9F/W21F/F222W mutant, however, exhibited no such pH-dependent changes. Time-resolved fluorescence anisotropy studies revealed a ligand-dependent, Tyr-9-dependent, change in the order parameter of Trp-222. However, no pH dependence was observed. In equilibrium and pre-steady-state ligand binding studies, product conjugate had a decreased equilibrium binding affinity (KD), concomitant with increased binding and dissociation rates, at higher pH values. Furthermore, the recovered pKa values for the pH-dependent microscopic rate constants ranged from 7.7 to 8.4, also in agreement with the pKa of Tyr-9. In contrast, the Y9F/W21F/F222W mutant had no pH-dependent transition in KD or rate constants for ligand binding or dissociation. The combined results indicate that the macroscopic populations of "open" and "closed" states of the C terminus are not determined solely by the ionization state of Tyr-9. However, the rates of transition between these states are faster for the ionized Tyr-9. The ionized Tyr-9 states provide a parallel pathway for product dissociation, which is kinetically and thermodynamically favored. In silico kinetic models further support the functional role for the parallel dissociation pathway provided by ionized Tyr-9.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610, USA.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Ibarra
authors.forename: Pramit
authors.initials: P
authors.lastname: Chowdhury
authors.forename: Jacob W
authors.initials: JW
authors.lastname: Petrich
authors.forename: William M
authors.initials: WM
authors.lastname: Atkins
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005454:Fluorescence Polarization; D015870:Gene Expression; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D014443:Tyrosine; D005982:Glutathione Transferase; D005978:Glutathione; C033550:hexylglutathione