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 pi-I-dependent properties. Aspartic proteases comprise an impor tant class of enzyme targets for structure-based drug design. We have performed a complete thermodynamic study of pepstatin binding to plasm epsin II, an aspartic proteinase found in Plasmodium falciparum, using isothermal titration calorimetry and circular dichroism. Thermodynami c parameters (Delta G, Delta H, Delta C-p, and Delta S) 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 so lvent and the complex. We used thermodynamic proton linkage theory to derive both the pH-independent binding energetics for pepstatin and th e number and pK(a), values of ionizable residues whose pK(a) values ch ange during ligand binding. These residues were identified as the two catalytic aspartates, with pK(a)s of 6.5 and 3.0, and His 164, with a pK(a) of 7.5, based on the three-dimensional structure of the pepstati n-plasmepsin IT complex. At pH 5.0, where the protease has optimum act ivity, the proton transfer process contributes almost 40% of the total binding free energy change and the total charge of the active-site as partic 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 str uctural data for pepstatin binding with human cathepsin D, a lysosomal aspartic protease that shares 35% sequence identity with plasmepsin I T, suggests that the energetic differences between these two proteins are due to a higher interdomain flexibility in plasmepsin II.