Localization of the C‐terminus of rat glutathione S‐transferase A1‐1: Crystal structure of mutants W21F and W21F/F220Y

Adman, Elinor T.; Trong, I. Le; Stenkamp, Ronald E.; Nieslanik, Brenda S.; Dietze, Eric C.; Tai, Guoying; Ibarra, Catherine; Atkins, William M.

doi:10.1002/1097-0134(20010201)42:2<192::aid-prot60>3.0.co;2-#

Cited by 28 publications

(46 citation statements)

References 34 publications

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“…The DSC and CD analyses clearly indicate that the low temperature asymmetry in the DSC scan is due mainly to the dynamic C-terminal helix of GSTA1-1, which becomes localized when ligand is bound. A greater degree of conformational heterogeneity for apo-GSTA1-1, compared with GSTA1-1 when the active-site is occupied, agrees well with previous crystallographic (45,46,50,51), NMR (47), and timeresolved fluorescence studies (12), and the data presented here. More interestingly, the DSC analysis based on modified Landau theory of phase transitions indicates that the low temperature asymmetry seen in the apo-GSTA1-1 DSC data is due to an increase in the number of accessible conformational substates accessible for sampling and that there are barrierless transitions between substates in the ensemble.…”

Section: Resultssupporting

confidence: 91%

“…To investigate this predenaturation asymmetry further, similar studies were completed with addition of saturating concentrations of the product analog, S-hexyl glutathione, which is known to cause the C terminus to adopt a well defined conformation (45)(46)(47). Interestingly, the low temperature asymmetry features of the DSC trace of apo-GSTA1-1 were eliminated when S-hexyl GSH was bound, and the thermogram retained only the main unfolding transition, as observed in the absence of ligand, albeit shifted to a higher temperature.…”

Section: Resultsmentioning

confidence: 99%

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Ensemble Perspective for Catalytic Promiscuity

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Acchione

Sumida

et al. 2011

Journal of Biological Chemistry

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Background: Catalytic promiscuity is common, but its molecular basis is poorly understood. Results: Differential scanning calorimetry reveals the local active site conformational landscape of a promiscuous detoxification enzyme, glutathione transferase A1-1, as "smooth" and heterogeneous. Conclusion: Facile conformational exchange facilitates substrate promiscuity. Significance: The results provide the first thermodynamic basis for catalytic promiscuity.

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Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Ensemble Perspective for Catalytic Promiscuity

Honaker

Acchione

Sumida

et al. 2011

Journal of Biological Chemistry

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“…The binding of S-hexylglutathione (S-hexyl-GSH) to GSTA1-1 has been shown to induce closure of the C terminus, with Tyr-9 pK a (ϳ9) remaining lower than the pK a of free tyrosine in solution (13,24,25). Here the emission spectra of W21F/F222W and Y9F/W21F/F222W were also obtained in the presence of saturating concentrations of S-hexyl-GSH.…”

Section: Resultsmentioning

confidence: 92%

The Anomalous pK of Tyr-9 in Glutathione S-Transferase A1-1 Catalyzes Product Release

Ibarra

Chowdhury

Petrich

et al. 2003

Journal of Biological Chemistry

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The pK a of the catalytic Tyr-9 in glutathione S-transferase (GST) A1-1 is lowered from 10.3 to ϳ8.1 in the apoenzyme and ϳ9.0 with a GSH conjugate bound at the active site. However, a clear functional role for the unusual Tyr-9 pK a has not been elucidated. GSTA1-1 also includes a dynamic C terminus that undergoes a liganddependent 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 pHdependent change in Trp-222 emission properties consistent with changes in C-terminal solvation or conformation. The apparent pK a 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 pK a 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 (K D ), concomitant with increased binding and dissociation rates, at higher pH values. Furthermore, the recovered pK a values for the pH-dependent microscopic rate constants ranged from 7.7 to 8.4, also in agreement with the pK a of Tyr-9. In contrast, the Y9F/ W21F/F222W mutant had no pH-dependent transition in K D 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.

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“…The conjugation of GSH with a foreign compound generally results in the formation of a nontoxic product that can be readily eliminated. 6 In addition to their catalytic function, GSTs also serve as nonenzymatic binding proteins, known as ligandins, that interact with various lipophilic compounds that include steroid and thyroid hormones. Finally, reactive oxygen species can trigger apoptosis, programmed cell death, in cells.…”

Section: Introductionmentioning

confidence: 99%

“…The production of GSTs, by helping to protect the cell from oxidative damage caused by reactive oxygen species, assists in obviating the induction of apoptosis. 6 Cytochrome P450, especially the form CYP3A4 which predominates in hepatic tissue, has functions similar to that of GST. It metabolizes endogenous compounds and xenobiotics.…”

Section: Introductionmentioning

confidence: 99%

Interaction of Glutathione S-Transferase with Hypericin: A Photophysical Study

et al. 2005

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The photophysics of hypericin have been studied in its complex with two different isoforms, A1-1 and P1-1, of the protein glutathione S-transferase (GST). One molecule of hypericin binds to each of the two GST subunits. Comparisons are made with our previous results for the hypericin/human serum albumin complex (Photochem. Photobiol. 1999, 69, 633−645). Hypericin binds with high affinity to the GSTs: 0.65 μM for the A1-1 isoform and 0.51 μM for the P1-1 isoform (Biochemistry 2004, 43, 12761−12769). The photophysics and activity of hypericin are strongly modulated by the binding protein. Intramolecular hydrogen-atom transfer is suppressed in both cases. Most importantly, while there is significant singlet oxygen generation from hypericin bound to GST A1-1, binding to GST P1-1 suppresses singlet oxygen generation to almost negligible levels. The data are rationalized in terms of a simple model in which the hypericin photophysics depends entirely upon the decay of the triplet state by two competing processes, quenching by oxygen to yield singlet oxygen and ionization, the latter of these two are proposed to be modulated by A1-1 and P1-1. The photophysics of hypericin have been studied in its complex with two different isoforms, A1-1 and P1-1, of the protein glutathione S-transferase (GST). One molecule of hypericin binds to each of the two GST subunits. Comparisons are made with our previous results for the hypericin/human serum albumin complex (Photochem. Photobiol. 1999, 69, 633-645). Hypericin binds with high affinity to the GSTs: 0.65 µM for the A1-1 isoform and 0.51 µM for the P1-1 isoform (Biochemistry 2004, 43, 12761-12769). The photophysics and activity of hypericin are strongly modulated by the binding protein. Intramolecular hydrogen-atom transfer is suppressed in both cases. Most importantly, while there is significant singlet oxygen generation from hypericin bound to GST A1-1, binding to GST P1-1 suppresses singlet oxygen generation to almost negligible levels. The data are rationalized in terms of a simple model in which the hypericin photophysics depends entirely upon the decay of the triplet state by two competing processes, quenching by oxygen to yield singlet oxygen and ionization, the latter of these two are proposed to be modulated by A1-1 and P1-1.

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Localization of the C‐terminus of rat glutathione S‐transferase A1‐1: Crystal structure of mutants W21F and W21F/F220Y

Cited by 28 publications

References 34 publications

Ensemble Perspective for Catalytic Promiscuity

Ensemble Perspective for Catalytic Promiscuity

The Anomalous pK of Tyr-9 in Glutathione S-Transferase A1-1 Catalyzes Product Release

Interaction of Glutathione S-Transferase with Hypericin: A Photophysical Study

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