The effect of the Y108V mutation of human glutathione S-transferase P1-1 (hGST P1-1) on the binding of the diuretic drug ethacrynic acid (EA) and its glutathione conjugate (EASG) was investigated by calorimetric, spectrofluorimetric, and crystallographic studies. The mutation Tyr 108 fi Val resulted in a 3D-structure very similar to the wild type (wt) enzyme, where both the hydrophobic ligand binding site (H-site) and glutathione binding site (G-site) are unchanged except for the mutation itself. However, due to a slight increase in the hydrophobicity of the H-site, as a consequence of the mutation, an increase in the entropy was observed. The Y108V mutation does not affect the affinity of EASG for the enzyme, which has a higher affinity (K d~0 .5 lM) when compared with those of the parent compounds, K EA d~1 3 lM, K GSH d~2 5 lM. The EA moiety of the conjugate binds in the H-site of Y108V mutant in a fashion completely different to those observed in the crystal structures of the EA or EASG wt complex structures. We further demonstrate that the DC p values of binding can also be correlated with the potential stacking interactions between Additional Supporting Information may be found in the online version of this article.Abbreviations: DMSO, dimethylsulfoxide; DSC, differential scanning calorimetry; DTT, dithiothreitol; EA, ethacrynic acid; EACys, ethacrynic-cysteine conjugate; EAME, ethacrynic-mercaptoethanol conjugate; EASG, ethacrynic-glutathione conjugate; GST, glutathione transferase; HEPES, N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid; hGST P1-1, human glutathione transferase P1-1; ITC, isothermal titration calorimetry; MES, 2-morpholinoethanesulfonic acid; MPD, 2-methyl-2,4-pentanediol; wt, wild type.Indalecio Quesada-Soriano and Lorien J. Parker contributed equally to this work.The coordinates for the structures of the Y108V apo, EA and EASG complex are deposited with the Protein Databank (http:// rcsg.org/pdb/) with the entry codes 3HJM, 3HKR, and 3HJO, respectively. ligand and residues located in the binding sites as predicted from crystal structures. Moreover, the mutation does not significantly affect the global stability of the enzyme. Our results demonstrate that calorimetric measurements maybe useful in determining the preference of binding (the binding mode) for a drug to a specific site of the enzyme, even in the absence of structural information.

The binding properties of a glutathione S-transferase (EC 2.5.1.18) from Schistosoma japonicum to substrate glutathione (GSH) has been investigated by intrinsic fluorescence and isothermal titration calorimetry (ITC) at pH 6.5 over a temperature range of 15±30 8C. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of GSH at pH 6.5. We have also studied the effect of pH on the thermodynamics of GSH±GST interaction. The behaviour shown at different pHs indicates that at least three groups must participate in the exchange of protons. Fluorimetric and calorimetric measurements indicate that GSH binds to two sites in the dimer of 26-kDa glutathione S-transferase from Schistosoma japonicum (SjGST). On the other hand, noncooperativity for substrate binding to SjGST was detected over a temperature range of 15±30 8C.Among thermodynamic parameters, whereas DG8 remains practically invariant as a function of temperature, DH and DS8 both decrease with an increase in temperature. While the binding is enthalpically favorable at all temperatures studied, at temperatures below 25 8C, DG8 is also favoured by entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attaining a value of zero at 24.3 8C, and then becoming unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy±entropy compensation. The temperature dependence of the enthalpy change yields the heat capacity change (DC p 8) of 20.238^0.04 kcal per K per mol of GSH bound.

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S-nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1-1 (hGSTP1-1) at 1.4 Å resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild-type hGSTP1-1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild-type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S-nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix a2 of hGST P1-1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild-type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix a2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S-nitrosylation of Cys47 by GSNO is a likely physiological scenario.Keywords: calorimetry; glutathione S-transferase; nitric oxide; nitrosoglutathione; X-ray crystallography Supplemental material: see www.proteinscience.org Glutathione transferases (EC 2.5.1.18; GSTs) are a superfamily of enzymes involved in the biotransformation of numerous carcinogenic, mutagenic, toxic, and pharmacologically active compounds (Jakoby and Habig 1980). The human cytosolic GSTs are dimeric proteins grouped into at least eight species-independent classes (a, k, m, p, v, s, u, and z) on the basis of their amino acid sequence, substrate specificity, and immunological properties (Mannervik et al. 1985;Meyer et al. 1991;Buetler and Eaton Reprint requests to: Luis García-Fuentes, Department of Physical Chemistry, Biochemistry and Inorganic Chemistry, Faculty of Experimental Sciences, University of Almería, La Cañ ada de San Urbano, 04120 Almería, Spain; e-mail: lgarcia@ual.es; fax: +34-950-015008.Abbreviations: DSC, differential scanning calorimetry; DTT, dithiothreitol; GSNO, S-nitrosoglutathione; GST, glutathione S-transferase; hGSTP1-1, human glutathione transferase P1-1; HEPES, N-(2-hydroxyethyl)-piperazine-N9-2-ethanesulfonic acid; ITC, isothermal titration calorimetry; MES, 2-morpholinoethanesulfonic acid; PEG, polyethylene glycol.Article published online ahead of print. Article and publication date are at

High-sensitivity titration calorimetry is used to measure changes in enthalpy, heat capacity and protonation for

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