Analysis of crystal structures of aspartic proteinases: on the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes

Abstract

To elucidate the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes, we analyzed and compared the crystal structures of these enzymes, their complexes with inhibitors, and zymogens in the active site area (a total of 82 structures). In addition to the water molecule (W1) located between the active carboxyls and playing a role of the nucleophile during catalytic reaction, another water molecule (W2) at the vicinity of the active groups was found to be completely conserved. This water molecule plays an essential role in formation of a chain of hydrogen-bonded residues between the active site flap and the active carboxyls on ligand binding. These data suggest a new approach to understanding the role of residues around the catalytic site, which can assist the development of the catalytic reaction. The influence of groups adjacent to the active carboxyls is manifested by pepsin activity at pH 1.0. Some features of pepsin-like enzymes and their mutants are discussed in the framework of the approach.

Fig. 1.

The catalytic site and the surrounding region of unbound porcine pepsin in monoclinic crystals (pdb code: 4pep; resolution 1.8 Å, Sielecki et al. 1990). In these crystals, the flap is involved in intermolecular contacts, and it is close to the molecular surface. The hydroxyl of Tyr 75 forms hydrogen bonds with the interior of the molecule, resulting in formation of a continuous chain of hydrogen-bonded residues Trp 39–Tyr 75–W2–Ser 35–Asp 32 extending from the flap to the active site. The same chain of hydrogen bonds is a characteristic property of all complexes of pepsin-like enzymes with inhibitors. The Thr 218 hydroxyl group, locating at a hydrogen-bond distance from the Asp 215 carboxyl, protects this carboxyl from protonation in acid media.

Fig. 2.

The conformation of Thr 218 residue in porcine pepsinogen (pdb code: 3psg; resolution 1.65 Å, Hartsuck et al. 1992). In pepsinogen, a hydrogen bond between Thr 218 and Asp 215 is not possible because Asp 215 is involved in hydrogen bonds with Lys 36 and Tyr 37 of the propart. The hydroxyl group of Thr 218 can form only a weak hydrogen bond with water molecule W540, although it is close to Ser 219 NH group. In this structure, Thr 218 residue is present as a rotamer unusual for active enzymes.

Fig. 3.

The catalytic site and the surrounding region of inhibited memapsin 2 by OM 99-2 inhibitor (pdb code: 1fkn; resolution 1.9 Å, Hong et al. 2000). The figure shows the conserved nature of the chain of hydrogen bonds connecting the flap with the active site, which forms on inhibitor binding. Trp 80(76) plays the role of Trp 39, replaced in memapsin by Ala.

Fig. 4.

The catalytic site and the surrounding region of active penicillopepsin (pdb code: 3app; resolution 1.8 Å, James and Sielecki 1983) representing the arrangement of residues in the active site area, when the flap is close to the open position. The flap does not form contacts with the Trp 39 O^ɛ1 atom or water molecule W2. At the same time, the distance between the Ser 35 O^γ atom and Asp 32 carboxyl is too large for the formation of a hydrogen bond.

Fig. 5.

(a) Scheme showing the arrangement of hydrogen bonds in the active site area of pepsin-like enzymes during formation of enzyme–substrate complex when Tyr 75 approaches Trp 39 and water molecule W2. At the same time, Ser 35 becomes close to the Asp 32 carboxyl. Trp 39 donates proton to the Tyr 75 hydroxyl and forces it to donate proton to water molecule W2. As a result, Ser 35 becomes a proton donor to the Asp 32 carboxyl, enhancing its acidic properties (Asp 32 presumes to leave its proton participating in formation of the gem-diol structure of the tetrahedral intermediate). Hydrogen-bond distances shown in both a and b are not relevant to any structure. In the scheme, they show only the presence of hydrogen bonds. (b) Scheme showing a possible reorientation of water molecule W2 after moving of Tyr 75 out of the interior of a molecule. Reorientation of the proton of the Ser 35 hydroxyl results in some increase of basic properties of the Asp 32 carboxyl, which helps to return its initial protonated state after the catalytic reaction.

Fig. 5.

(a) Scheme showing the arrangement of hydrogen bonds in the active site area of pepsin-like enzymes during formation of enzyme–substrate complex when Tyr 75 approaches Trp 39 and water molecule W2. At the same time, Ser 35 becomes close to the Asp 32 carboxyl. Trp 39 donates proton to the Tyr 75 hydroxyl and forces it to donate proton to water molecule W2. As a result, Ser 35 becomes a proton donor to the Asp 32 carboxyl, enhancing its acidic properties (Asp 32 presumes to leave its proton participating in formation of the gem-diol structure of the tetrahedral intermediate). Hydrogen-bond distances shown in both a and b are not relevant to any structure. In the scheme, they show only the presence of hydrogen bonds. (b) Scheme showing a possible reorientation of water molecule W2 after moving of Tyr 75 out of the interior of a molecule. Reorientation of the proton of the Ser 35 hydroxyl results in some increase of basic properties of the Asp 32 carboxyl, which helps to return its initial protonated state after the catalytic reaction.

Fig. 6.

Formula representation of the two steps of the catalytic reaction, showing the interaction of the catalytic groups with the surrounding residues. (a) The initial step before substrate binding. (b) Tetrahedral intermediate (Davies 1990; Parris et al. 1992; James et al. 1992; Veerapandian et al. 1992) and its interaction with groups, adjacent to the catalytic site. We suppose the hydrogen bond of the Asp 215 carboxyl with the Thr 218 carboxyl is weakened after substrate binding, while the bond of the Asp 32 carboxyl with the Ser 35 hydroxyl becomes stronger.