title: 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.
authors.affiliation: Structural Biochemistry Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, USA. xie@ncifcrf.gov
authors.forename: D
authors.initials: D
authors.lastname: Xie
authors.forename: S
authors.initials: S
authors.lastname: Gulnik
authors.forename: L
authors.initials: L
authors.lastname: Collins
authors.forename: E
authors.initials: E
authors.lastname: Gustchina
authors.forename: L
authors.initials: L
authors.lastname: Suvorov
authors.forename: J W
authors.initials: JW
authors.lastname: Erickson
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D002151:Calorimetry; D002402:Cathepsin D / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010436:Pepstatins / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology*; D011480:Protease Inhibitors / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011522:Protons; D015800:Protozoan Proteins; D013816:Thermodynamics
chemical_list: D010436:Pepstatins; D011480:Protease Inhibitors; D011522:Protons; D015800:Protozoan Proteins; C014195:Streptomyces pepsin inhibitor; D016282:Aspartic Acid Endopeptidases; C100637:plasmepsin II; D002402:Cathepsin D; C031375:pepstatin