Proton Linkage Research Articles

Intrinsic thermodynamics of 4-substituted-2,3,5,6-tetrafluorobenzenesulfonamide binding to carbonic anhydrases by isothermal titration calorimetry

Para substituted tetrafluorobenzenesulfonamides bind to carbonic anhydrases (CAs) extremely tightly and exhibit some of the strongest known protein–small ligand interactions, reaching an intrinsic affinity of 2 pM as determined by displacement isothermal titration calorimetry (ITC). The enthalpy and entropy of binding to five CA isoforms were measured by ITC in two buffers of different protonation enthalpies. The pKa values of compound sulfonamide groups were measured potentiometrically and spectrophotometrically, and enthalpies of protonation were measured by ITC in order to evaluate the proton linkage contributions to the observed binding thermodynamics. Intrinsic means the affinity of a sulfonamide anion for the Zn bound water form of CAs. Fluorination of the benzene ring significantly enhanced the observed affinities as it increased the fraction of deprotonated ligand while having little impact on intrinsic affinities. Intrinsic enthalpy contributions to the binding affinity were dominant over entropy and were more exothermic for CA I than for other CA isoforms. Thermodynamic measurements together with the X-ray crystallographic structures of protein–ligand complexes enabled analysis of structure–activity relationships in this enzyme ligand system.

The differences in binding 12‐carbon aliphatic ligands by bovine β‐lactoglobulin isoform A and B studied by isothermal titration calorimetry and X‐ray crystallography

Isoforms A (LGB-A) and B (LGB-B) of bovine lactoglobulin, the milk protein, differ in positions 64 (D↔G) and 118 (V↔A). Interactions of LGB-A and LGB-B with sodium dodecyl sulfate (SDS), dodecyltrimethylammonium chloride (DTAC) and lauric acid (LA), 12-carbon ligands possessing differently charged polar groups, were investigated using isothermal titration calorimetry and X-ray crystallography, to study the proton linkage phenomenon and to distinguish between effects related to different isoforms and different ligand properties. The determined values of ΔS and ΔH revealed that for all ligands, binding is entropically driven. The contribution from enthalpy change is lower and shows strong dependence on type of buffer that indicates proton release from the protein varying with protein isoform and ligand type and involvement of LA and Asp64 (in isoform A) in this process. The ligand affinities for both isoforms were arranged in the same order, DTAC < LA < SDS, and were systematically lower for variant B. The entropy change of the complexation process was always higher for isoform A, but these values were compensated by changes in enthalpy, resulting in almost identical ΔG for complexes of both isoforms. The determined crystal structures showed that substitution in positions 64 and 118 did not influence the overall structure of LGB complexes. The chemical character of the ligand polar group did not affect the position of its aliphatic chain in protein β-barrel, indicating a major role of hydrophobic interactions in ligand binding that prevailed even with the repulsion between positively charged DTAC and lysine residues located at binding site entrance.

Strategies for assessing proton linkage to bimolecular interactions by global analysis of isothermal titration calorimetry data

Isothermal titration calorimetry (ITC) is a traditional and powerful method for studying the linkage of ligand binding to proton uptake or release. The theoretical framework has been developed for more than two decades and numerous applications have appeared. In the current work, we explored strategic aspects of experimental design. To this end, we simulated families of ITC data sets that embed different strategies with regard to the number of experiments, range of experimental pH, buffer ionization enthalpy, and temperature. We then re-analyzed the families of data sets in the context of global analysis, employing a proton linkage binding model implemented in the global data analysis platform SEDPHAT, and examined the information content of all data sets by a detailed statistical error analysis of the parameter estimates. In particular, we studied the impact of different assumptions about the knowledge of the exact concentrations of the components, which in practice presents an experimental limitation for many systems. For example, the uncertainty in concentration may reflect imperfectly known extinction coefficients and stock concentrations or may account for different extents of partial inactivation when working with proteins at different pH values. Our results show that the global analysis can yield reliable estimates of the thermodynamic parameters for intrinsic binding and protonation, and that in the context of the global analysis the exact molecular component concentrations may not be required. Additionally, a comparison of data from different experimental strategies illustrates the benefit of conducting experiments at a range of temperatures.

Effects of Proton Linkage on Thermodynamic Properties of Enzyme– Antibiotic Complexes of the Aminoglycoside Nucleotidyltransferase (2″)-Ia

Aminoglycoside nucleotidyltransferase(2″)-Ia (ANT) catalyzes the covalent modification of certain aminoglycoside antibiotics and imparts resistance to bacteria which possess this enzyme. Isothermal titration calorimetry (ITC) experiments using two structurally similar substrates (kanamycin A and kanamycin B) and a structurally different inhibitor (neomycin) were performed to determine pH and proton linkage effects on thermodynamic properties of enzyme–aminoglycoside complexes. Data showed that there was a decrease in pKa for one or more carboxyl groups on the enzyme that interacts with 2′-amino group of neomycin or kanamycin B. Protonation state of this site exerted a significant effect on the binding affinity of ligand to ANT. ITC experiments also showed that the loss of affinity at higher pH values was not as pronounced for neomycin compared to kanamycins. This difference is a result of higher pK a s for free neomycin which are up shifted in the ANT–neomycin complex. The decrease in affinity and differences in binding linked protonation above and below the pH optimum cannot be fully explained by these two factors. Therefore, the formation of ANT–aminoglycoside complexes involves protonation and deprotonation of multiple functional groups on both ligand and enzyme which also contribute to the pH profile of the complex formation.

Osmotic enhancement of DNA binding by glutamate: Klenow and Klentaq DNA binding studies

Glutamate (Glu−) is the major osmolyte accumulated in E.coli in response to external salt. In vitro, the 'glutamate effect' is primarily characterized by enhanced DNA binding when Glu− replaces Cl−. Some proteins also exhibit reduced ion release upon DNA binding in Glu−. Klenow polymerase (E.coli) exhibits both aspects of the 'glutamate effect'. Upon replacement of Cl− by Glu−, the ion release upon DNA binding is reduced by &gt;50%. In addition, the apparent water release upon DNA binding is reduced by ~70% (150 waters) and proton linkage increases 5 fold. The decrease in apparent water release highlights the osmotic nature of the glutamate effect: Glu− and Cl− salts behave as ionic inhibitors of DNA binding but Glu− salts also behave as osmotic enhancers. While Klentaq's (T.aquaticus) DNA binding is enhanced in Glu−, its ionic and proton linkages are not altered. The osmotic enhancement is present for Klentaq polymerase but it is not as significant in the salt concentration range at which nanomolar Klentaq‐DNA binding occurs. E.coli DNA‐binding proteins might have evolved to bind tightly at higher salt concentrations in order to use osmotic enhancement of Glu− upon its intracellular accumulation. NSF.

Klenow and Klentaq-DNA Binding: the ‘Glutamate Effect’ is Primarily an Osmotic Effect

DNA binding by Klenow (E.coli) and Klentaq (T.aquaticus) DNA polymerases has been studied as a function of monovalent salt concentration, pH and osmotic stress. We previously showed that DNA binding resulted in the net release of 4.5∼5 ions from Klenow and 3∼3.5 ions from Klentaq. Here, we report that Klenow and Klentaq have minimal sensitivity to pH changes, with proton linkages of ∼0.06 and ∼0.3 respectively in KCl. Furthermore, osmotic stress data in KCl indicates 500∼600 waters are released upon binding by both polymerases.Glutamate is the major intracellular anion accumulated in E.coli in the presence of KCl in the external environment. The ‘glutamate effect’ is primarily characterized by an increase in DNA binding affinity when chloride is replaced by glutamate. Some proteins also exhibit decreased ionic linkage in glutamate.Klenow exhibits both aspects of the ‘glutamate effect’. Substituting glutamate for chloride reduces the ionic linkage for Klenow by >50%. The presence of glutamate also increases the proton linkage of Klenow five fold and decreases water release by ∼70% to approximately 150 waters. The dramatic decrease in water release highlights the osmotic nature of the glutamate effect. Glutamate and chloride salts behave as ionic inhibitors of DNA binding but glutamate salts also exhibit an osmotic enhancement effect.While Klentaq's DNA binding affinity is also enhanced by glutamate, its ionic and proton linkages are not altered. The osmotic enhancement is present for Klentaq but it is not as significant in the salt concentration range at which nanomolar Klentaq-DNA binding occurs. E.coli DNA-binding proteins might have evolved to bind tightly at higher salt concentration to utilize the glutamate effect while accumulating intracellular glutamate.

Thermodynamic Investigation of the Role of Contact Residues of β-Lactamase-inhibitory Protein for Binding to TEM-1 β-Lactamase

We have determined the thermodynamics of binding for the interaction between TEM-1 beta-lactamase and a set of alanine substituted contact residue mutants ofbeta-lactamase-inhibitory protein (BLIP) using isothermal titration calorimetry. The binding enthalpies for these interactions are highly temperature dependent, with negative binding heat capacity changes ranging from -800 to -271 cal mol(-1) K(-1). The isoenthalpic temperatures (at which the binding enthalpy is zero) of these interactions range from 5 to 38 degrees C. The changes in isoenthalpic temperature were used as an indicator of the changes in enthalpy and entropy driving forces, which in turn are related to hydrophobic and hydrophilic interactions. A contact residue of BLIP is categorized as a canonical residue if its alanine substitution mutant exhibits a change of isoenthalpic temperature matching the change of hydrophobicity because of the mutation. A contact position exhibiting a change in isoenthalpic temperature that does not match the change in hydrophobicity is categorized as an anti-canonical residue. Our experimental results reveal that the majority of residues where alanine substitution results in a loss of affinity are canonical (7 of 10), and about half of the residues where alanine substitutions have a minor effect are canonical. The interactions between TEM-1beta-lactamase and BLIP canonical contact residues contribute directly to binding free energy, suggesting potential anchoring sites for binding partners. The anti-canonical behavior of certain residues may be the result of mutation-induced modifications such as structural rearrangements affecting contact residue configurations. Structural inspection of BLIP suggests that the Lys(74) side chain electrostatically holds BLIP loop 2 in position to bind to TEM-1 beta-lactamase, explaining a large loss of entropy-driven binding energy of the K74A mutant and the resulting anti-canonical behavior. The anti-canonical behavior of the W150A mutant may also be due to structural rearrangements. Finally, the affinity enhancing effect of the contact residue mutant Y50A may be due to energetic coupling interactions between Asp(49) and His(41).

Proton exchange coupled to the specific binding of alkylsulfonates to serum albumins

We have applied isothermal titration calorimetry to investigate the linkage between ligand binding and the uptake or release of protons by human serum albumin (HSA) and bovine serum albumin (BSA). The ligands were sodium decyl sulfate (SDeS) and sodium dodecyl sulfate (SDS). Within a certain temperature range, the binding isotherm could be clearly resolved into two classes of sites (high affinity and low affinity) and modeled assuming independence and thermodynamic equivalence of the sites within each class. Measurements at pH 7.0 in different buffer systems revealed that the binding of SDS to the high affinity sites did not couple to any exchange of protons in either of the proteins. Saturation of the 6–8 low affinity sites for SDS, on the other hand, brought about the release of two protons from both HSA and BSA. In addition to elucidating the pH dependence of ligand binding, this analysis stressed that binding enthalpies for the low affinity sites measured by calorimetry must be corrected for effects due to the concomitant protonation of the buffer. The shorter ligand SDeS bound to HSA with a comparable stoichiometry but with four times lower affinity. Interestingly, no proton linkage was observed for the binding of SDeS. An empirical structural analysis suggested that His 242 in site 7 (of HSA) is a likely candidate for one of the proton donors.

Proton Linkage for CO Binding and Redox Properties of Bovine Lactoperoxidase

The pH-dependence of redox properties and of CO binding to bovine lactoperoxidase has been investigated over the range between 2 and 11. The pH-dependence of redox potentials shows a biphasic behavior, suggesting the existence of (at least) two redox-linked groups, which change their pK a values upon reduction. These values are in close agreement with those observed to play a relevant role in the modulation of CO binding to ferrous bovine lactoperoxidase. They have been tentatively attributed to Arg-372 and His-226, which are located on the distal side of the heme pocket of lactoperoxidase. A complete and unequivocal description of the proton-linked behavior of bovine lactoperoxidase requires, however, three residues, which are redox linked and relevant for the modulation of CO binding. The rate constant for CO binding to bovine lactoperoxidase is slower than what is reported for most hemoproteins, suggesting that these two residues, Arg-372 and His-226, are representing a severe barrier for the access of exogenous ligands to the heme. This aspect has been further investigated by fast kinetics following laser photolysis, trying to obtain information on the ligand binding pathway and on the energy barriers.

Thermodynamic analysis of binding and protonation in DOTAP/DOPE (1:1): DNA complexes using isothermal titration calorimetry

A better understanding of the nature of the interaction between various cationic lipids used for gene delivery and DNA would lend insight into their structural and physical properties that may modulate their efficacy. We therefore separated the protonation and binding events which occur upon complexation of 1:1 DOTAP (1,2-dioleoyl-3-trimethylammonium propane):DOPE (1,2-dioleoylphosphatidylethanolamine) liposomes to DNA using proton linkage theory and isothermal titration calorimetry (ITC). The enthalpy of DOPE protonation was estimated as −45.0±0.7 kJ/mol and the intrinsic binding enthalpy of lipid to DNA as +2.8±0.3 kJ/mol. The p K a of DOPE was calculated to shift from 7.7±0.1 in the free state to 8.8±0.1 in the complex. At physiological ionic strength, proton linkage was not observed upon complex formation and the buffer-independent binding enthalpy was +1.0±0.4 kJ/mol. These studies indicate that the intrinsic interaction between 1:1 DOTAP/DOPE and DNA is an entropy-driven process and that the affinities of cationic lipids that are formulated with and without DOPE for DNA are controlled by the positive entropic changes that occur upon complex formation.

Driving Forces of Protein Association: The Dimer-Octamer Equilibrium in Arylsulfatase A

The enzyme arylsulfatase A (ASA) occurs in solution as dimer ( α 2) above pH 6 and associates to octamers ( α 2) 4 below pH 6. The crystal structure of ASA suggests that the ( α 2)-( α 2) 4 equilibrium is regulated by protonation/deprotonation of Glu-424 located at the interface between ( α 2) dimers in the octamer. The reason for this assumption is that Glu-424 can be in two different conformers where it forms an intra or intermolecular hydrogen bond, respectively. In the present study we investigate this protein association process theoretically. The electrostatic energies are evaluated by solving the Poisson-Boltzmann equation for the inhomogeneous dielectric of the protein-water system for the dimer and octamer configurations. If a conventional surface energy term is used for the nonelectrostatic interactions, the absolute value of free energy of association fails to agree with experiment. A more detailed treatment that explicitly accounts for hydrophilic and hydrophobic character of the amino acids in the dimer-dimer interface of the octamer can explain this discrepancy qualitatively. The pH dependence of the computed association energy clearly demonstrates that the octamer is more stable at low pH if Glu-424 becomes protonated and forms an intermolecular hydrogen bond. We found a slight preference of Glu-424 to be in a conformation where its acidic group is fully solvent-exposed in the dimer state to form hydrogen bonds with water molecules. Application of the proton linkage model to calculate the association energy from the simulated data yielded results identical to the one obtained from the corresponding direct method.

Thermodynamic characterization of the interaction of human cyclophilin 18 with cyclosporin A

Isothermal titration calorimetry (ITC) was used to investigate thermodynamic parameters of the cyclosporin A (CsA)-cyclophilin 18 (hCyp18) association reaction. We have calculated the thermodynamic parameters (enthalpy, entropy, heat capacity, and free energy of binding) of the CsA/hCyp18 complexation. All but two methods described in the literature underestimate the affinity to hCyp18 of CsA. We found that the association constant (1.1·108 M−1 at 10 °C) of CsA to hCyp18 is in close agreement with the reciprocal of the reported inhibitory constant of the peptidylprolyl cis/trans isomerase activity of hCyp18. Interpretation of the thermodynamic parameters in buffered solution of water, 30% glycerol and D2O leads to the conclusion that the highly specific binding of CsA to hCyp18 is mainly mediated through hydrogen bonding and to a lesser degree through hydrophobic interaction. Furthermore, the pH dependence of the association constant was determined and analyzed according to a single proton linkage model, resulting in a pKa value of 5.7 in free hCyp18 and below 4.5 in the CsA complexed form. Titration experiments using different single component buffers possessing different heats of ionization allowed us to estimate that statistically half a proton is transferred upon CsA binding from the binding interface of hCyp18 to the buffer at pH 5.5. No proton transfer was detected at pH 7.5. The thermodynamic results are discussed in relation to the published X-ray and NMR structure of the free and CsA complexed hCyp18.

Experimental and Calculated Shift in pKa upon Binding of Phosphotyrosine Peptide to the SH2 Domain of p56lck

The pH dependence of the affinity of a 11-mer phosphotyrosine (pY) peptide (EPQpYEEIPIYL-NH2) for the SH2 domain of the tyrosine kinase p56lck was investigated with surface plasmon resonance (SPR). From SPR competition experiments the affinity in solution was obtained. The pH dependence of the affinity in solution can be well described by a proton linkage model with a single pKa shift upon binding, from 6.1 to 4.7. This shift is ascribed to the transition from the −2 to the −1 ionisation state of the tyrosine phosphate group. Based on the X-ray structure for the complex with Lck SH2, a pKa value of 5.3 for the bound pY peptide was computed, modelling the solvated protein as a system of point charges in a continuum. With the phosphate in the −2 state the binding energy is 1.8 kcal/mol more favourable than for the −1 state, corresponding to a 20-fold higher affinity. A proper charge is relevant in the design of potential therapeutic Lck SH2 ligands with mimics for the metabolically unstable tyrosine phosphate group.

Contribution of proton linkage to the thermodynamic stability of the major cold-shock protein of Escherichia coli CspA.

The stability of protein is defined not only by the hydrogen bonding, hydrophobic effect, van der Waals interactions, and salt bridges. Additional, much more subtle contributions to protein stability can arise from surface residues that change their properties upon unfolding. The recombinant major cold shock protein of Escherichia coli CspA an all-beta protein unfolds reversible in a two-state manner, and behaves in all other respects as typical globular protein. However, the enthalpy of CspA unfolding strongly depends on the pH and buffer composition. Detailed analysis of the unfolding enthalpies as a function of pH and buffers with different heats of ionization shows that CspA unfolding in the pH range 5.5-9.0 is linked to protonation of an amino group. This amino group appears to be the N-terminal alpha-amino group of the CspA molecule. It undergoes a 1.6 U shift in pKa values between native and unfolded states. Although this shift in pKa is expected to contribute approximately 5 kJ/mol to CspA stabilization energy the experimentally observed stabilization is only approximately 1 kJ/mol. This discrepancy is related to a strong enthalpy-entropy compensation due, most likely, to the differences in hydration of the protonated and deprotonated forms of the alpha-amino group.

Comparison of binding energies of SrcSH2-phosphotyrosyl peptides with structure-based prediction using surface area based empirical parameterization.

The prediction of binding energies from the three-dimensional (3D) structure of a protein-ligand complex is an important goal of biophysics and structural biology. Here, we critically assess the use of empirical, solvent-accessible surface area-based calculations for the prediction of the binding of Src-SH2 domain with a series of tyrosyl phosphopeptides based on the high-affinity ligand from the hamster middle T antigen (hmT), where the residue in the pY+ 3 position has been changed. Two other peptides based on the C-terminal regulatory site of the Src protein and the platelet-derived growth factor receptor (PDGFR) are also investigated. Here, we take into account the effects of proton linkage on binding, and test five different surface area-based models that include different treatments for the contributions to conformational change and protein solvation. These differences relate to the treatment of conformational flexibility in the peptide ligand and the inclusion of proximal ordered solvent molecules in the surface area calculations. This allowed the calculation of a range of thermodynamic state functions (deltaCp, deltaS, deltaH, and deltaG) directly from structure. Comparison with the experimentally derived data shows little agreement for the interaction of SrcSH2 domain and the range of tyrosyl phosphopeptides. Furthermore, the adoption of the different models to treat conformational change and solvation has a dramatic effect on the calculated thermodynamic functions, making the predicted binding energies highly model dependent. While empirical, solvent-accessible surface area based calculations are becoming widely adopted to interpret thermodynamic data, this study highlights potential problems with application and interpretation of this type of approach. There is undoubtedly some agreement between predicted and experimentally determined thermodynamic parameters: however, the tolerance of this approach is not sufficient to make it ubiquitously applicable.

Thermodynamic dissection of the binding energetics of KNI-272, a potent HIV-1 protease inhibitor.

KNI-272 is a powerful HIV-1 protease inhibitor with a reported inhibition constant in the picomolar range. In this paper, a complete experimental dissection of the thermodynamic forces that define the binding affinity of this inhibitor to the wild-type and drug-resistant mutant V82F/184V is presented. Unlike other protease inhibitors, KNI-272 binds to the protease with a favorable binding enthalpy. The origin of the favorable binding enthalpy has been traced to the coupling of the binding reaction to the burial of six water molecules. These bound water molecules, previously identified by NMR studies, optimize the atomic packing at the inhibitor/protein interface enhancing van der Waals and other favorable interactions. These interactions offset the unfavorable enthalpy usually associated with the binding of hydrophobic molecules. The association constant to the drug resistant mutant is 100-500 times weaker. The decrease in binding affinity corresponds to an increase in the Gibbs energy of binding of 3-3.5 kcal/mol, which originates from less favorable enthalpy (1.7 kcal/mol more positive) and entropy changes. Calorimetric binding experiments performed as a function of pH and utilizing buffers with different ionization enthalpies have permitted the dissection of proton linkage effects. According to these experiments, the binding of the inhibitor is linked to the protonation/deprotonation of two groups. In the uncomplexed form these groups have pKs of 6.0 and 4.8, and become 6.6 and 2.9 in the complex. These groups have been identified as one of the aspartates in the catalytic aspartyl dyad in the protease and the isoquinoline nitrogen in the inhibitor molecule. The binding affinity is maximal between pH 5 and pH 6. At those pH values the affinity is close to 6 x 10(10) M(-1) (Kd = 16 pM). Global analysis of the data yield a buffer- and pH-independent binding enthalpy of -6.3 kcal/mol. Under conditions in which the exchange of protons is zero, the Gibbs energy of binding is -14.7 kcal/mol from which a binding entropy of 28 cal/K mol is obtained. Thus, the binding of KNI-272 is both enthalpically and entropically favorable. The structure-based thermodynamic analysis indicates that the allophenylnorstatine nucleus of KNI-272 provides an important scaffold for the design of inhibitors that are less susceptible to resistant mutations.

Structural energetics of serine protease inhibition

Abstract

Proton linkage of cytochrome a oxidoreduction in carbon monoxide-treated cytochrome c oxidase

Oxidoreduction of the low spin haem a of cytochrome c oxidase was recently reported to be coupled to release/uptake of nearly one proton from/to the enzyme at pH 7.5 in the presence of CO to block oxidoreduction of the binuclear haem a 3/Cu B centre (N. Capitanio et al., Biochim. Biophys. Acta, 1318 (1997) 255–265). This is difficult to reconcile with earlier findings from several laboratories that the pH-dependence of the E m of haem a is ca. 10 mV/pH unit over a wide pH range in such conditions, which implies redox coupling of only ca. 0.17 H +/e −. In order to resolve this discrepancy, we have performed careful measurements of proton release coupled to oxidation of haem a and Cu A in CO-inhibited cytochrome aa 3 from bovine heart mitochondria. We find that oxidation of these centres by ferricyanide leads to release of a total of 0.20 protons per enzyme molecule at pH 7.7, increasing to 0.43 protons at pH 6.6, far short of a full 1 H +/e −. Using vesicles reconstituted with cytochrome c oxidase, we also found that all this proton release occurs towards the outside of the vesicles. The observed dependence can be explained by a model in which oxidoreduction of haem a is coupled to uptake and release of ca. 0.17 H +/e −, while oxidoreduction of Cu A is linked to a protonatable group which has a p K a of 6.2 when Cu A is in the reduced state. In agreement with existing data, this model predicts that the E m of Cu A will only be slightly pH dependent in the pH range of these measurements.

Calorimetric investigation of proton linkage by monitoring both the enthalpy and association constant of binding: application to the interaction of the Src SH2 domain with a high-affinity tyrosyl phosphopeptide.

The binding of Src homology 2 (SH2) domains to tyrosyl phosphopeptides depends on electrostatic interactions between the phosphotyrosine and its binding site. To probe the role of these interactions, we have used isothermal titration calorimetry to study the pH dependence of the binding of the SH2 domain of the Src kinase to a high-affinity tyrosyl phosphopeptide. Two independent approaches were employed. In a first series of experiments that focused on determining the peptide's association constant between pH 5.0 and 9.0, two ionizable groups were characterized. One group, with free and bound pKas of 6.2 and 4.4, respectively, could be identified as the phosphate in the phosphotyrosine while the other group, with free and bound pKas of 8.2 and 8.5, respectively, could be only tentatively assigned to a cysteine in the phosphotyrosine binding pocket. Further information on the linkage between peptide binding and protonation of the phosphotyrosine was obtained from a second series of experiments, which focused on determining the peptide binding enthalpy at low values of pH in several buffers with different ionization enthalpies. These data provided free and bound pKa values for the phosphotyrosine identical to those derived from the first series of experiments, and hence demonstrated for the first time that the two approaches provide identical information regarding proton linkage. In addition, the second series of experiments also determined the intrinsic enthalpy of binding of both the protonated and deprotonated phosphate forms of the peptide. These two sets of experiments provided a complete energetic profile of the linkage between phosphate ionization and peptide binding. From this profile, it was determined that the PO32- form of the peptide binds 2.3 kcal mol-1 more favorably than the PO3H1- form due entirely to a more favorable entropy of binding.

Dissection of the pH dependence of inhibitor binding energetics for an aspartic protease: direct measurement of the protonation states of the catalytic aspartic acid residues.

The catalytic activity and inhibitor binding energetics of enzymes are often pH-dependent properties. Aspartic proteases comprise an important class of enzyme targets for structure-based drug design. We have performed a complete thermodynamic study of pepstatin binding to plasmepsin II, an aspartic proteinase found in Plasmodium falciparum, using isothermal titration calorimetry and circular dichroism. Thermodynamic parameters (DeltaG, DeltaH, DeltaCp, and DeltaS) were measured as functions of both pH and temperature. In the pH range from 4.5 to 7.0, pepstatin binding is accompanied by proton transfer between the solvent and the complex. We used thermodynamic proton linkage theory to derive both the pH-independent binding energetics for pepstatin and the number and pKa values of ionizable residues whose pKa values change during ligand binding. These residues were identified as the two catalytic aspartates, with pKas of 6.5 and 3.0, and His 164, with a pKa of 7.5, based on the three-dimensional structure of the pepstatin-plasmepsin II complex. At pH 5.0, where the protease has optimum activity, the proton transfer process contributes almost 40% of the total binding free energy change and the total charge of the active-site aspartic acid residues is -1. These experimental results provide direct measurement for the protonation states of the catalytic aspartates in the presence of bound ligands. Comparison of the thermodynamic and structural data for pepstatin binding with human cathepsin D, a lysosomal aspartic protease that shares 35% sequence identity with plasmepsin II, suggests that the energetic differences between these two proteins are due to a higher interdomain flexibility in plasmepsin II.