A thermodynamic characterization of the interaction of 8-anilino-1-naphthalenesulfonic acid with native globular proteins: the effect of the ligand dimerization in the analysis of the binding isotherms.

8-Anilino-1-naphthalenesulfonic acid (ANS) is a popular fluorescence probe, broadly used for the analysis of proteins, but the nature of its interaction with proteins and the high increase in the fluorescence intensity that takes place upon such process are still unclear. In the last few years, isothermal titration calorimetry has been used to characterize the nature of the interaction of this dye with proteins. The analysis of the binding isotherms of these studies has not considered the dimerization equilibrium of ANS, which is pH dependent, and it can result in serious errors in the data analysis. In the present work we have developed a suitable data analysis by which this process is taken into account. To study the binding of the dye to proteins at different pH values, we have used the Abl-SH3 domain. Our results suggest that at pH 3 and 5, where the dimerization of the ANS is important, electrostatic interactions are significant for the binding of ANS to the Abl-SH3 domain. However, at pH 7, ANS behaves mostly as monomer and the interaction with the protein is mainly hydrophobic. The pH dependent behavior of the ANS binding to proteins can be explained in terms of ionization states of both, the protein and the ANS.

Thermodynamic determination of the binding constants of angiotensin-converting enzyme inhibitors by a displacement method.

Somatic angiotensin I-converting enzyme (s-ACE) plays a central role in blood pressure regulation and has been the target of most antihypertensive drugs. A displacement isothermal titration calorimetry method has been used to accurately determine the binding constant of three strong s-ACE inhibitors. Under the experimental conditions studied in this work, the relative potency of the inhibitors was determined to be enalaprilat>lisinopril>captopril. We analyze the thermodynamic behaviour of the binding process using the new structural information provided by the ACE structures, as well as the conformational changes that occur upon binding.

Site-directed mutagenesis indicates an important role of cysteines 76 and 181 in the catalysis of hydantoin racemase from Sinorhizobium meliloti.

Hydantoin racemase enzyme plays a crucial role in the reaction cascade known as "hydantoinase process." In conjunction with a stereoselective hydantoinase and a stereospecific carbamoylase, it allows the total conversion from D,L-5-monosubstituted hydantoins, with a low rate of racemization, to optically pure D- or L-amino acids. Residues Cys76 and Cys181 belonging to hydantoin racemase from Sinorhizobium meliloti (SmeHyuA) have been proved to be involved in catalysis. Here, we report biophysical data of SmeHyuA Cys76 and Cys181 to alanine mutants, which point toward a two-base mechanism for the racemization of 5-monosubstituted hydantoins. The secondary and the tertiary structure of the mutants were not significantly affected, as shown by circular dichroism. Calorimetric and fluorescence experiments have shown that Cys76 is responsible for recognition and proton retrieval of D-isomers, while Cys181 is responsible for L-isomer recognition and racemization. This recognition process is further supported by measurements of protein stability followed by chemical denaturation in the presence of the corresponding compound.

Kinetic analysis and modelling of the allosteric behaviour of liver and muscle glycogen phosphorylases.

Allosteric enzymes have very complex kinetic behaviours which are primarily interpreted through simplified models. To describe the functional properties of liver and muscle glycogen phosphorylase isozymes we have developed an experimental strategy based on the measurements of initial reaction rates in the presence of different concentrations of the effectors glucose-1-phosphate and methyl-xanthines. Using the extensive structural information available for the two glycogen phosphorylase conformers T (inactive) and R (active) with different ligands, we have applied the Monod-Wyman-Changeux model and analysed the results in the context of the exclusive binding of the inhibitors to the T state, meanwhile the substrate glucose-1-phosphate binds to both, the R and T states. The kinetic analysis shows a good agreement between our model and the results obtained from the glycogen phosphorylases and inhibitors included in this study, which demonstrates the validity of the approach described here.

Thermodynamic and mutational studies of l-N-carbamoylase from Sinorhizobium meliloti CECT 4114 catalytic centre.

Purified site-directed mutants of Sinorhizobium meliloti CECT 4114 l-N-carbamoylase (SmLcar) in which Glu132, His230, Asn279 and Arg292 were replaced have been studied by kinetic methods and isothermal titration calorimetry (ITC). The importance of His230, Asn279 and Arg292 residues in the recognition of N-carbamoyl-l-alpha-amino acids has been proved. The role of Glu132 has been confirmed in substrate hydrolysis. ITC has confirmed two Ni atoms per monomer of wild type enzyme, and two equal and independent substrate binding sites (one per monomer). Homology modelling of SmLcar supports the importance of His87, His194, His386, Glu133 and Asp98 in metal binding. A comprehensive reaction mechanism is proposed on the basis of binding experiments measured by ITC, kinetic assays, and homology of the active centre with beta-alanine synthase from Saccharomyces kluyveri and other enzymes.

Enzymatic activity assay of D-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates.

Isothermal titration calorimetry (ITC) has been applied to the determination of the activity of D-hydantoinase (EC 3.5.2.2) with several substrates by monitoring the heat released during the reaction. The method is based on the proportionality between the reaction rate and the thermal power (heat/time) generated. Microcalorimetric assays carried out at different temperatures provided the dependence of the catalytic rate constant on temperature. We show that ITC assay is a nondestructive method that allows the determination of the catalytic rate constant (kcat), Michaelis constant (KM), activation energy and activation Gibbs energy, enthalpy and entropy of this reaction.

Binding studies of hydantoin racemase from Sinorhizobium meliloti by calorimetric and fluorescence analysis.

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific d-carbamoylase guarantee the total conversion from d,l-5-monosubstituted hydantoins with a low velocity of racemization, to optically pure d-amino acids. Hydantoin racemase from Sinorhizobium meliloti was expressed in Escherichia coli. Calorimetric and fluorescence experiments were then carried out to obtain the thermodynamic binding parameters, deltaG, deltaH and DeltaS for the inhibitors L- and D-5-methylthioethyl-hydantoin. The number of active sites is four per enzyme molecule (one per monomer), and the binding of the inhibitor is entropically and enthalpically favoured under the experimental conditions studied. In order to obtain information about amino acids involved in the active site, four different mutants were developed in which cysteines 76 and 181 were mutated to Alanine and Serine. Their behaviour shows that these cysteines are essential for enzyme activity, but only cysteine 76 affects the binding to these inhibitors.

Crystallographic and thermodynamic analysis of the binding of S-octylglutathione to the Tyr 7 to Phe mutant of glutathione S-transferase from Schistosoma japonicum.

Glutathione S-transferases are a family of multifunctional enzymes involved in the metabolism of drugs and xenobiotics. Two tyrosine residues, Tyr 7 and Tyr 111, in the active site of the enzyme play an important role in the binding and catalysis of substrate ligands. The crystal structures of Schistosoma japonicum glutathione S-transferase tyrosine 7 to phenylalanine mutant [SjGST(Y7F)] in complex with the substrate glutathione (GSH) and the competitive inhibitor S-octylglutathione (S-octyl-GSH) have been obtained. These new structural data combined with fluorescence spectroscopy and thermodynamic data, obtained by means of isothermal titration calorimetry, allow for detailed characterization of the ligand-binding process. The binding of S-octyl-GSH to SjGST(Y7F) is enthalpically and entropically driven at temperatures below 30 degrees C. The stoichiometry of the binding is one molecule of S-octyl-GSH per mutant dimer, whereas shorter alkyl derivatives bind with a stoichiometry of two molecules per mutant dimer. The SjGST(Y7F).GSH structure showed no major structural differences compared to the wild-type enzyme. In contrast, the structure of SjGST(Y7F).S-octyl-GSH showed asymmetric binding of S-octyl-GSH. This lack of symmetry is reflected in the lower symmetry space group of the SjGST(Y7F).S-octyl-GSH crystals (P6(3)) compared to that of the SjGST(Y7F).GSH crystals (P6(3)22). Moreover, the binding of S-octyl-GSH to the A subunit is accompanied by conformational changes that may be responsible for the lack of binding to the B subunit.

A monomer form of the glutathione S-transferase Y7F mutant from Schistosoma japonicum at acidic pH.

Dissociation and unfolding of homodimeric glutathione S-transferase Y7F mutant from Schistosoma japonicum (SjGST-Y7F) were investigated at equilibrium using urea as denaturant. The conserved residue Tyr7 plays a central role in the catalytic mechanism and the mutation Tyr-Phe yields an inactive enzyme that is able to bind the substrate GSH with a higher binding constant than the wild type enzyme. Mutant SjGST-Y7F is a dimer at pH 6 or higher and a stable monomer at pH 5 that binds GSH (K value of 1.2x10(5)+/-6.4x10(3)M(-1) at pH 6.5 and 6.3x10(4)+/-1.25x10(3)M(-1) at pH 5). The stability of the SjGST-Y7F mutant was studied by urea induced unfolding techniques (DeltaG(W)=13.86+/-0.63kcalmol(-1) at pH 6.5 and DeltaG(W)=11.22+/-0.25kcalmol(-1) at pH 5) and the monomeric form characterized by means of size exclusion chromatography, fluorescence, and electrophoretic techniques.

Thermodynamics of glutathione binding to the tyrosine 7 to phenylalanine mutant of glutathione S-transferase from Schistosoma japonicum.

The binding of glutathione (GSH) to the tyrosine 7 to phenylalanine mutant of Schistosoma japonicum glutathione S-transferase (SjGST-Y7F) has been studied by isothermal titration calorimetry (ITC). At pH 6.5 and 25 degrees C this mutant shows a higher affinity for glutathione than wild type enzyme despite an almost complete loss of activity in the presence of 1-chloro-2,4-dinitrobenzene (CDNB) as second substrate. The enthalpy change upon binding of GSH is more negative for the mutant than for the wild type GST (SjGST). Changes in accessible solvent areas (ASA) have been calculated based on enthalpy and heat capacity changes. ASA values indicated the burial of apolar surfaces of protein and ligand upon binding. A more negative DeltaC(p) value has been obtained for the mutant enzyme, suggesting a more hydrophobic interaction, as may be expected from the change of a tyrosine residue to phenylalanine.