Aspartic protease-pepstatin A interactions: Structural insights on the thermal inactivation mechanism.

Aspartic proteases are the targets for structure-based drug design for their role in physiological processes
and pharmaceutical applications. Structural insights into the thermal inactivation mechanism of an
aspartic protease in presence and absence of bound pepstatin A have been obtained by kinetics of
thermal inactivation, CD, fluorescence spectroscopy and molecular dynamic simulations. The irreversible
thermal inactivation of the aspartic protease comprised of loss of tertiary and secondary structures
succeeded by the loss of activity, autolysis and aggregation The enthalpy and entropy of thermal inactivation
of the enzyme in presence of pepstatin A increased from 81.2 to 148.5 kcal mol�1, and from 179
to 359 kcal mol�1 K�1 respectively. Pepstatin A shifted the mid-point of thermal inactivation of the
protease from 58 �C to 77 �C. The association constant (K) for pepstatin A with aspartic protease was
2.5 ± 0.3 � 10 5 M�1 and DGo value was �8.3 kcal mol�1. Molecular dynamic simulation studies were able
to delineate the role of pepstatin A in stabilizing backbone conformation and side chain interactions. In
the Ca-backbone, the short helical segments and the conserved glycines were part of the most unstable
segments of the protein. Understanding the mechanism of thermal inactivation has the potential to
develop re-engineered thermostable proteases.