       Document 0321
 DOCN  M9610321
 TI    Thermodynamic mapping of the inhibitor site of the aspartic protease
       endothiapepsin.
 DT    9601
 AU    Gomez J; Freire E; Department of Biology, Johns Hopkins University,
       Baltimore, MD; 21218, USA.
 SO    J Mol Biol. 1995 Sep 22;252(3):337-50. Unique Identifier : AIDSLINE
       MED/96004886
 AB    The discovery that the protease from the human immunodeficiency virus
       (HIV) belongs to the aspartic protease family has generated renewed
       interest in this class of proteins. In this paper, the interactions of
       endothiapepsin, an aspartic proteinase from the fungus Endothia
       parasitica, with the inhibitor pepstatin A have been studied by
       high-sensitivity calorimetric techniques. These experiments have
       permitted a complete characterization of the temperature and
       pH-dependence of the binding energetics. The binding reaction is
       characterized by negative intrinsic binding enthalpy and negative heat
       capacity changes. The association constant is maximal at low pH (2 x
       10(9) M-1 at pH 3) but decreases upon increasing pH (8.1 x 10(6) M-1 at
       pH 7). The binding of the inhibitor is coupled to the protonation of one
       of the aspartic moieties in the Asp dyad of the catalytic site of the
       protein. This phenomenon is responsible for the decrease in the apparent
       affinity of the inhibitor for the enzyme upon increasing pH. The
       experimental results presented here indicate that the binding of the
       inhibitor is favored both enthalpically and entropically. While the
       favorable enthalpic contribution is intuitively expected, the favorable
       entropic contribution is due to the large gain in solvent-related
       entropy associated with the burial of a large hydrophobic surface, that
       overcompensates the loss in conformational and translational/rotational
       degrees of freedom upon complex formation. The characteristics of the
       molecular recognition process have been evaluated by means of
       structure-based thermodynamic analysis. Three regions in the protein
       contribute significantly to the free energy of binding: the residues
       surrounding the Asp dyad (Asp32 in the N-terminal lobe and Asp215 in the
       C-terminal domain) and the flap region (Ile73 to Asp77). In addition,
       the rearrangement of residues that are not in immediate contact with the
       inhibitor provides close to 40% of the protease contribution to the
       binding free energy. On the other hand, the two statine residues provide
       more than half of the inhibitor contributions to the total free energy
       of binding. It is demonstrated that a previously developed empirical
       structural parametrization of the thermodynamic parameters that define
       the Gibbs energy, accurately accounts for the binding energetics and its
       temperature and pH-dependence.
 DE    Aspartic Proteinases/*ANTAGONISTS & INHIB/*CHEMISTRY  Binding Sites
       Calorimetry, Differential Scanning/METHODS  Comparative Study
       Hydrogen-Ion Concentration  Kinetics  Models, Molecular  Models,
       Structural  Pepstatins/*PHARMACOLOGY  Protease Inhibitors/*PHARMACOLOGY
       *Protein Structure, Secondary  Sensitivity and Specificity
       Sphaeriales/ENZYMOLOGY  Support, U.S. Gov't, P.H.S.  Thermodynamics
       JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

