Carmen Barón

Calorimetric and structural studies of the nitric oxide carrier S-nitrosoglutathione bound to human glutathione transferase P1-1

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 α2 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 α2 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.

Enthalpy of captopril‐angiotensin I‐converting enzyme binding

High‐sensitivity titration calorimetry is used to measure changes in enthalpy, heat capacity and protonation for the binding of captopril to the angiotensin I‐converting enzyme (ACE; EC 3.4.15.1). The affinity of ACE to captopril is high and changes slightly with the pH, because the number of protons linked to binding is low. The determination of the enthalpy change at different pH values suggests that the protonated group in the captopril‐ACE complex exhibits a heat protonation of approximately −30 kJ/mol. This value agrees with the protonation of an imidazole group. The residues which may become protonated in the complex could be two histidines existing in two active sites, which are joined to the amino acids coordinated to Zn2+. Calorimetric measurements indicate that captopril binds to two sites in the monomer of ACE, this binding being enthalpically unfavorable and being dominated by a large positive entropy change. Thus, binding is favored by both electrostatic and hydrophobic interactions. The temperature dependence of the free energy of binding ΔG° is weak because of the enthalpy‐entropy compensation caused by a large heat capacity change, ΔC p=−4.3±0.1 kJ/K/mol of monomeric ACE. The strong favorable binding entropy and the negative ΔC p indicate both a large contribution to binding due to hydrophobic effects, which seem to originate from dehydration of the ligand‐protein interface, and slight conformational changes in the vicinity of the active sites.

A calorimetric study of the binding of S-alkylglutathiones to glutathione S-transferase

The binding of three competitive glutathione analogue inhibitors (S-alkylglutathione derivatives) to glutathione S-transferase from Schistosoma japonicum, SjGST, has been investigated by isothermal titration microcalorimetry at pH 6.5 over a temperature range of 15--30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that no protons are exchanged during the binding of S-alkylglutathione derivatives. Thus, at pH 6.5, the protons released during the binding of substrate may be from its thiol group. Calorimetric analyses show that S-methyl-, S-butyl-, and S-octylglutathione bind to two equal and independent sites in the dimer of SjGST. The affinity of these inhibitors to SjGST is greater as the number of methylene groups in the hydrocarbon side chain increases. In all cases studied, Delta G(0) remains invariant as a function of temperature, while Delta H(b) and Delta S(0) both decrease as the temperature increases. The binding of three S-alkylglutathione derivatives to the enzyme is enthalpically favourable at all temperatures studied. The temperature dependence of the enthalpy change yields negative heat capacity changes, which become less negative as the length of the side chain increases.

Calorimetric analysis of lisinopril binding to angiotensin I-converting enzyme.

Isothermal titration microcalorimetry has been used to measure changes in enthalpy and heat capacity for binding of lisinopril to the angiotensin I‐converting enzyme (ACE; EC 3.4.15.1) and to its apoenzyme at pH 7.5 over a temperature range of 15–30°C. Calorimetric measurements indicate that lisinopril binds to two sites in the monomer of both holo‐ and apo‐ACE. Binding of lisinopril to both systems is enthalpically unfavorable and, thus, is dominated by a large positive entropy change. The enthalpy change of binding is strongly temperature‐dependent for both holo‐ and apo‐ACE, arising from a large heat capacity change of binding equal to −2.4±0.2 kJ/K/(mol of monomeric holo‐ACE) and to −1.9±0.2 kJ/K/(mol of monomeric apo‐ACE), respectively. The negative values of ΔCp for both systems are consistent with burial of a large non‐polar surface area upon binding. Although the binding of lisinopril to holo‐ and apo‐ACE is favored by entropy changes, this is more positive for the holoenzyme. Thus, the interaction between Zn2+ and lisinopril results in a higher affinity of the holoenzyme for this drug due to a more favorable entropic contribution.

Influence of the H-site residue 108 on human glutathione transferase P1-1 ligand binding: Structure-thermodynamic relationships and thermal stability

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 → 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 (Kd ∼ 0.5 μM) when compared with those of the parent compounds, K  dEA ∼ 13 μM, K  dGSH ∼ 25 μM. 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 ΔCp values of binding can also be correlated with the potential stacking interactions between 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.

Thermodynamic Characterization of 5′-AMP Binding to Bovine Liver Glycogen Phosphorylase a

The binding of adenosine 5′-monophosphate to liver glycogen phosphorylase a (EC 2.4.1.1) has been studied by size exclusion high performance liquid chromatography and isothermal titration microcalorimetry at pH 6.9 over a temperature range of 25 to 35°C. The results are compared with those of the binding of the same nucleotide to the muscle isozyme and to liver phosphorylase b. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of the nucleotide. The dimer of liver glycogen phosphorylase a has been shown to have two equal and independent sites for 5′-AMP, which would correspond to the activator sites identified in the muscle isozyme. The binding constants as well as the changes in Gibbs energy, enthalpy, and entropy per site for 5′-AMP binding were calculated at each temperature. The results show that the major contribution to the negative value of ΔG0 stems from the value of ΔH in the range of 25 to 35°C. The enthalpy change of binding is strongly temperature-dependent, arising from a large negative ΔCp of binding equal to −1.45 ± 0.02 kJ K−1 (mol of 5′-AMP bound)−1, which suggests significant changes in the polar and apolar surfaces accessible to the solvent.

Identifying and characterizing binding sites on the irreversible inhibition of human glutathione S-transferase P1-1 by S-thiocarbamoylation

Human glutathione S‐transferase P1‐1 (hGST P1‐1) is involved in cell detoxification processes through the conjugation of its natural substrate, reduced glutathione (GSH), with xenobiotics. GSTs are known to be overexpressed in tumors, and naturally occurring isothiocyanates, such as benzyl isothiocyanate (BITC), are effective cancer chemopreventive compounds. To identify and characterize the potential inhibitory mechanisms of GST P1‐1 induced by isothiocyanate conjugates, we studied the binding of GST P1‐1 and some cysteine mutants to the BITC–SG conjugate as well as to the synthetic S‐(N‐benzylcarbamoylmethyl)glutathione conjugate (BC–SG). We report here the inactivation of GST P1‐1 through the covalent modification of two Cys47 residues per dimer and one Cys101. The evidence has been compiled by isothermal titration calorimetry (ITC) and electrospray ionization mass spectrometry (ESI‐MS). ITC experiments suggest that the BITC–SG conjugate generates adducts with Cys47 and Cys101 at physiological temperatures through a corresponding kinetic process, in which the BITC moiety is covalently bound to these enzyme cysteines through an S‐thiocarbamoylation reaction. ESI‐MS analysis of the BITC–SG incubated enzymes indicates that although the Cys47 in each subunit is covalently attached to the BITC ligand moiety, only one of the Cys101 residues in the dimer is so attached. A plausible mechanism is given for the emergence of inactivation through the kinetic processes with both cysteines. Likewise, our molecular docking simulations suggest that steric hindrance is the reason why only one Cys101 per dimer is covalently modified by BITC–SG. No covalent inactivation of GST P1‐1 with the BC–SG inhibitor has been observed. The affinities and inhibitory potencies for both conjugates are high and very similar, but slightly lower for BC–SG. Thus, we conclude that the presence of the sulfur atom from the isothiocyanate moiety in BITC–SG is crucial for its irreversible inhibition of GST P1‐1.

Role of mutation Y6F on the binding properties of Schistosoma japonicum glutathione S-transferase

The role of the hydroxyl group of tyrosine 6 in the binding of Schistosoma japonicum glutathione S-transferase has been investigated by isothermal titration calorimetry (ITC). A site-specific replacement of this residue with phenylalanine produces the Y6F mutant, which shows negative cooperativity for the binding of reduced glutathione (GSH). Calorimetric measurements indicated that the binding of GSH to Y6F dimer is enthalpically driven over the temperature range investigated. A concomitant net uptake of protons upon binding of GSH to Y6F mutant was detected carrying out calorimetric experiments in various buffer systems with different heats of ionization. The entropy change is favorable at temperatures below 26 degrees C for the first site, being entropically favorable at all temperatures studied for the second site. The enthalpy change of binding is strongly temperature-dependent, arising from a large negative DeltaC(o) (p1)=-3.45+/-0.62kJK(-1)mol(-1) for the first site, whereas a small DeltaC(o) (p2)=-0.33+/-0.05kJK(-1)mol(-1) for the second site was obtained. This large heat capacity change is indicative of conformational changes during the binding of substrate.

Thermodynamic characterization of the binding of dCMP to the Asn229Asp mutant of thymidylate synthase

Isothermal titration microcalorimetry and equilibrium dialysis have been used to characterize the binding of 2′‐deoxycytidine 5′‐monophosphate (dCMP) to the Asn229Asp mutant of Lactobacillus casei recombinant thymidylate synthase at pH 7.4 over a temperature range of 15°C to 35°C. Equilibrium dialysis analysis shows that dCMP binds to two sites in the dimer of both wild‐type and mutant thymidylate synthase. A concomitant net uptake of protons with binding of dCMP to both enzymes, was detected carrying out calorimetric experiments in various buffer systems with different heats of ionization. The change in protonation for binding of dCMP to wild‐type enzyme is lower than that obtained for binding of this nucleotide to TS N229D, which suggests that the pK value of Asp‐229 is increased upon dCMP binding to the mutant enzyme. At 25°C, although the binding of dCMP to wild‐type and N229D TS is favoured by both enthalpy and entropy changes, the enthalpy change is more negative for the mutant protein. Thus, the substitution of Asn 229 for Asp results in a higher affinity of TS for dCMP due to a more favourable enthalpic contribution. The Gibbs energy change of binding of dCMP to the mutant enzyme is weakly temperature‐dependent, because of the enthalpy‐entropy compensation arising from a negative heat capacity change of binding equal to −0.83±0.02 kJ K−1 per mol of dCMP bound.

Asn112 in Plasmodium falciparum glutathione S-transferase is essential for induced reversible tetramerization by phosphate or pyrophosphate

The glutathione S-transferase from Plasmodium falciparum presents distinct features which are absent from mammalian GST isoenzyme counterparts. Most apparent among these are the ability to tetramerize and the presence of a flexible loop. The loop, situated between the 113-119 residues, has been reported necessary for the tetramerization process. In this article, we report that a residue outside of this loop, Asn112, is a key to the process - to the point where the single Asn112Leu mutation prevents tetramerization altogether. We propose that a structural pattern involving the interaction of the Asn112 and Lys117 residues from two neighboring subunits plays a role in keeping the tetramer structure stable. We also report that, for the tetramerization of the wild-type PfGST to occur, phosphate or pyrophosphate anions must be present. In other words, tetramerization is a phosphate- or pyrophosphate-induced process. Furthermore, the presence of magnesium reinforces this induction. We present experimental evidence for these claims as well as a preliminary calorimetric and kinetic study of the dimeric Asn112Leu PfGST mutant. We also propose a putative binding site for phosphate or pyrophosphate anions through a comparative structural analysis of PfGST and pyrophosphatases from several organisms. Our results highlight the differences between PfGST and the human isoenzymes, which make the parasite enzyme a suitable antimalarial target.