Endothiapepsin from Endothia parasitica
is a member of the aspartic proteinase class of enzymes that are widely distributed among fungi, plants, vertebrates and viruses. A defining feature of this class is inhibition by the microbial peptide pepstatin, which contains the unusual amino acid statine (Bailey et al.
). In the HIV retrovirus, the proteinase (PR) is essential for maturation of the virus particle (Cooper, 2002
) and PR inhibitors have a proven therapeutic record in the treatment of AIDS. Aspartic proteinases also play major roles in hypertension, amyloid disease and malaria and have been implicated in tumorigenesis; inhibitors are much sought after as potential therapeutic agents. Aspartic proteinases comprise two structurally similar domains or subunits, each of which contributes an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond (Blundell et al.
). The fold of endothiapepsin is mainly β-sheet with small areas of α-helix on the outside of the protein; this fold is typical of all aspartic proteinases (Blundell et al.
Like most aspartic proteinases, endothiapepsin has an optimal acidic pH (4.5) and cleaves protein substrates with a similar specificity to that of porcine pepsin A. Two catalytic Asp-Thr-Gly sequences are conserved in these enzymes (Pearl & Blundell, 1984
), forming the active site, in which the carboxyl groups of the aspartates are held coplanar by a network of hydrogen bonds involving the surrounding main-chain and conserved amino-acid side-chain groups. A solvent molecule bound tightly to both carboxyls by hydrogen bonds is found in all native aspartic proteinase crystal structures. This water is within hydrogen-bonding distance of all four carboxyl O atoms and has been implicated in catalysis. Suguna et al.
) have suggested that it becomes partly displaced on substrate binding and is polarized by one of the aspartate carboxyls. The water then nucleophilically attacks the scissile-bond carbonyl group. The failure of numerous chemical studies to trap a covalently bound substrate supports the notion of a reaction mechanism that involves a noncovalently bound transition-state intermediate (Hofmann et al.
Short oligopeptide inhibitors of aspartic proteinases bind in β-strand conformations in the active-site cleft between the two domains, which is around 30 Å in length. The best synthetic inhibitors are those that mimic one or both of the hydroxyls in the putative transition state. One hydroxyl occupies the same position as the water molecule in the native enzyme and binds via
hydrogen bonds to both the catalytic aspartates (Bailey et al.
). Most of the transition-state analogues, such as statine-based inhibitors, mimic this hydroxyl group alone. Three X-ray structures of endothiapepsin complexed with this class of inhibitor have been refined to atomic resolution (Coates et al.
) and reveal a number of very short potential hydrogen bonds within the active site. In contrast, fluoroketone-analogue inhibitors [—CO—CF2
—] such as PD-135,040 (Fig. 1) mimic both hydroxyls in the putative intermediate, as they readily hydrate to the gem
-diol form [—C(OH)2
—]. Initial NMR studies using a ketone analogue of the scissile peptide bond suggested that it binds to the enzyme in a hydrated gem
-diol form [—C(OH)2
—] (Rich et al.
). Current mechanistic proposals have been based on the X-ray structures of gem
-diol transition-state analogues of the difluoroketone class [—C(OH)2
—] (Veerapandian et al.
; James et al.
; Coates et al.
). These structures have been refined at resolutions between 1.37 and 2.3 Å. The mechanism based on the structure of a fluoroketone as proposed by Veerapandian et al.
) is shown in Fig. 2. Early catalytic mechanisms differed in the assignment of the charges on the active-site aspartates.
The chemical structure of PD-135,040 in the hydrated gem-diol form.
Figure 2 The catalytic mechanism proposed by Veerapandian et al. (1992 ), which is based on the X-ray structure of a difluoroketone (gem-diol) inhibitor bound to endothiapepsin. A water molecule tightly bound to the aspartates in the native enzyme is (more ...)
Owing to the low pH optimum of the enzyme (Fruton, 1976
) and the proximity of the aspartates within the active site, it is likely that only one of the catalytic carboxyls will be charged at the optimal pH (4.5). The structure of the active site with a single hydroxyl transition-state analogue inhibitor bound allows only two possible positions for a proton on the catalytic aspartates: the inner O atom of Asp32 or the outer O atom of Asp215. A proton in either of the other two positions, i.e.
the outer O atom of Asp32 or the inner O atom of Asp215, would not have good enough geometry to form hydrogen bonds to the inhibitor hydroxyl. A neutron diffraction study of endothiapepsin complexed with the hydroxyethylene inhibitor H261 (Coates et al.
) showed that the outer O atom of Asp215 was protonated with the inhibitor bound, suggesting that Asp32 is negatively charged in the transition-state complex. The inhibitor H261 mimics one of the two hydroxyls thought to exist in the transition state. Until recently, it has not been possible to grow large protein crystals of endothiapepsin with any gem
-diol inhibitor that are suitable for neutron diffraction.
Here, we report the preparation of D2O-exchanged crystals of endothiapepsin complexed with a gem-diol inhibitor that are suitable for neutron data collection. We also report preliminary X-ray and neutron data collection at room temperature and X-ray data collection to ultrahigh resolution at low temperature. These studies provide the prospect of locating the catalytic H atoms in a complex that will give an unprecedented view of the tetrahedral intermediate.