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^,

Abstract

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.

Human cathepsin D.

“…Multifunctional catalysis is based on simultaneous effects of nucleophylic and electrophylic groups of the enzyme catalytic site on the carbonyl group of the peptide bond. Attachment of the substrate is followed by two synchronized transfer reactions: in the first, the proton is transferred from a water molecule onto a carboxyl anion of Asp33, in the other, the proton derived from a carboxyl group of Asp231 is transferred onto the oxygen of the carbonyl group of the substrate, giving rise to a tetraedric intermediate [81][82][83][84][85][86].…”

Interactions between trypsin and its peptidic inhibitors studied by isothermal titration calorimetry (ITC)

“…This finding is consistent with the data acquired from the crystallographic study [5] which showed that the interaction between native SFTI-1 and trypsin is stabilized by an extensive network of hydrogen . This approach has been applied earlier to several protein systems including aspartic protease [21], stromelysin-1 [22] and HIV-1 protease [23] to dissect protonation contributions from binding. The enthalpy measured in the ITC experiment, D ITC H, is the sum of all energetic effects accompanying the reaction, i.e.…”

Tracing Changes in Protonation: A Prerequisite to Factorize Thermodynamic Data of Inhibitor Binding to Aldose Reductase

“…for elastase, endothiapepsin, and plasmepsin II. 10,[43][44][45] Nevertheless, the present system experiences solubility limitations that restrict the required broad range pH variations. Furthermore, significant pH changes will likely alter the protonation states of other protein and ligand titratable groups, along with their ionization enthalpies in the bound and unbound state.…”