The
aspartic proteinases are an important family of
enzymes associated with several pathological conditions such as
hypertension (
renin),
gastric ulcers (
pepsin), neoplastic disease (
cathepsins D and E), and
AIDS (
HIV proteinase). Studies of inhibitor binding are therefore of great importance for design of novel inhibitors for potential therapeutic applications. Numerous X-ray analyses have shown that transition-state isostere inhibitors of
aspartic proteinases bind in similar extended conformations in the active-site cleft of the target
enzyme. Upon comparison of 21
endothiapepsin inhibitor complexes, the hydrogen bond lengths were found to be shortest where the isostere (P1-P'1) interacts with the
enzyme's catalytic
aspartate pair. Hydrogen bonds with good geometry also occur at
P'2, and more so at P3, where a conserved water molecule is involved in the interactions. Weaker interactions also occur at P2, where the side-chain conformations of the inhibitors appear to be more variable than at the more tightly held positions. At P2 and, to a lesser extent, P3, the side-chain conformations depend intriguingly on interactions with spatially adjacent side chains, namely P'1 and P1, respectively. The tight binding at P1-P'1, P3, and
P'2 is also reflected in the larger number of van der Waals contacts and the large decreases in
solvent-accessible area at these positions, as well as their low temperature factors. Our analysis substantiates earlier proposals for the locations of
protons in the transition-state complex.
Aspartate 32 is probably ionized in the complexes, its charge being stabilized by 1, or sometimes 2, hydrogen bonds from the transition-state analogues at P1. The detailed comparison also indicates that the P1 and P2 residues of substrate in the ES complex may be strained by the extensive binding interactions at P3, P'1, and
P'2 in a manner that would facilitate hydrolysis of the scissile
peptide bond.