Prolyl Endopeptidase
Created by: Sarah Smith
Prolyl endopeptidase (pdb: 1H2W) from Sus scrofa is a cytosolic serine protease which specifically cleaves the endo- side of prolyl residues within biologically active oligopeptides. Because proline residues are unique in that they are imino rather than amino acids, peptides that contain proline residues are often resistant to enzymatic degradation. Therefore, proline-specific enzymes like prolyl endopeptidase are necessary for the biological processing and degradation of peptide hormones and neuropeptides which contain proline. Prolyl endopeptidase has been found in most organisms and tissues, but may be the peptidase enzyme with the highest concentration in the brain; it is thus implicated in numerous neurological disorders, including depression, anorexia, bulimia nervosa, mania, schizophrenia, and Alzheimer’s disease. Because of these associations, prolyl endopeptidase is an important drug target (1).
Porcine prolyl endopeptidase was crystallized using the hanging drop vapor diffusion method. For this, the protein mixture (10mg/mL) was mixed with the mother liquor (“17% (w/v) methoxy-polyethylene glycol (MPEG) 5000, 15% (v/v) glycerol, 1% (v/v) monothioglycerol, 20 mM Ca(Ac)2, and 100 mM TRIS (pH 8.5)”) in a 1:1 ratio. This was left to equilibrate at 4oC and after four days, crystals appeared. The crystallized structure of prolyl endopeptidase contains a monothioglycerol ligand, which was used as a reducing agent during crystallization (2).
The activity of prolyl endopeptidase is dependent on its structure. Prolyl endopeptidase contains two domains: the N-terminal β-propeller domain, composed of residues 73-427, and the active site-containing C-terminal α/β-hydrolase domain, composed of residues 1-72 and 428-710 (3). The β-propellor domain contains 29 β-strands arranged in anti-parallel sheets that form a seven-bladed propeller tertiary structure. The α/β-hydrolase domain contains ten α-helices and ten β-strands, in both parallel and anti-parallel arrangements. The entire enzyme contains twelve instances of random coil. Several interdomain hydrogen bonds serve to stabilize the interactions of the two domains, including a hydrogen bond network between Thr-204, Hist-593, and Lys-588 predicted by modeling simulations and seen in crystallized prolyl endopeptidase. According to simulations with a bound Z-prolyl-prolinal substrate, five dominant hydrogen bonds were present between the two prolyl endopeptidase domains-those between Lys-458 and Asp-434, Gln-439 and Asp-356, Glu-512 and Pro-309, and a double hydrogen bond between Arg-643 and Asp 149 (4). Inter-domain salt bridges also exist between Asp-35 and Lys-196 and between Asp-149 and Arg-643 (5).
Unlike other members of the prolyl oligopeptidase family, prolyl endopeptidase does not have a cavity that could serve as a path for substrate entry and product release at the interface of its two domains, indicating that large structural changes are required for catalysis (6, 7). Studies have shown that the two domains of prolyl endopeptidase move, allowing the enzyme to oscillate between open and closed conformations. Although porcine prolyl endopeptidase has not been crystallized in both conformations, the opened and closed conformations of Aeromonas punctata (pdb IDs: 3IUL and 3IVM, respectively) have been determined and published (8). This oscillation is necessary to catalysis, as studies that have engineered a disulfide bridge cross-linking the two domains kept the prolyl endopeptidase in a permanently closed state and inactivated its peptidase activity (9).
The N-terminal domain of prolyl endopeptidase acts as a gate to exclude large substrates, making prolyl endopepitdase specifically an oligopeptidase. Both inter-domain and intra-domain motions are necessary for substrate entry and exit, and concerted action of the propeller blades and two domains has been suggested as the mechanism for substrate entry to the binding pocket (3). Several residues have been identified as lining the binding pocket, most notably Trp-595, which ring stacks with the prolyl residue of the substrate, providing stabilization of the enzyme-substrate complex (2, 4) and Arg-643 which hydrogen bonds with a carbonyl carbon of the substrate (4). The hydroxyl group of Tyr-473 and the peptide backbone amino group of Asn-555 hydrogen bond with the oxyanion of the tetrahedral intermediates formed in enzyme catalysis, providing further binding stabilization (2, 4, 10).
The cleavage of peptides by prolyl endopeptidase takes place via the catalytic triad, composed of Ser-554, His-680, and Asp-641 through an acylation-deacylation mechanism (Figure 1). In this process, the substrate enters the binding pocket of the enzyme, and then Asp-641 forms a hydrogen bond with His-680, immobilizing and orienting the histidine. His-680 then acts as a general base and removes a proton from Ser-554, increasing the nucleophilicity of Ser-554. Acylation occurs when Ser-554 attacks the carbonyl carbon of the prolyl residue, forming a tetrahedral intermediate enzyme-substrate complex. The tetrahedral intermediate then breaks down, cleaving the peptide. His-680 again acts as a general base and deprotonates a water molecule, which acts as a nucleophile and attacks the carbonyl carbon of the acyl enzyme. This creates a second tetrahedral intermediate, which collapses to deacylate the enzyme with assistance from proton donation from His-680 to Ser-554 to regenerate the resting state enzyme (1, 11-14).
Because of prolyl endopeptidase’s association with many neurological disorders, many inhibitors of this enzyme have been designed. Many of these are competitive inhibitors which contain a five-membered heterocylic ring at the position occupied by proline in natural substrates. Z-pro-prolinal (pdb ID: 1QFS) is one such competitive inhibitor. These inhibitors covalently bind to the enzyme, forming a tetrahedral intermediate like that formed in the reaction with natural substrates. However, as there is no good leaving group except the catalytic serine, the enzyme cannot be recycled. Additionally inhibitors form hydrogen bonds with residues in the oxyanion-binding site and do not freely dissociate, preventing substrate from binding. Racys et. al. showed that a five member heterocycle ring was not a necessary component of prolyl endopeptidase inhibitors by synthesizing an unnatural piperidine-containing substrate, compound two (pdb ID: 2XDW), which covalently bonds with Ser-554 and is stabilized by ring stacking with Trp-595. As with natural substrates, Tyr-473 and Asn-555 form hydrogen bonds with the inhibitor compound two (15). A class of inhibitors comprised of IC-1 (pdb ID: 4AMY), IC-3 (pdb ID= 4ANO), and IC-4 (pdb ID: 4AN1), has been designed based on the structure of Z-pro-prolinal. IC-2 (pdb ID: 4AMZ) mimics the proline structure of Z-pro-prolinal with a double bond of cyclopentane. IC-1, IC-2, and IC-4 bond covalently to the active site, forming a hemi-ketal, whereas IC-3 instead forms an iminoether bond. IC-4 has entered clinical trials under the name JTP-4819, and unlike Z-pro-prolinal, which can only accept hydrogen bonds, it can also donate to hydrogen bonds via an amide group (4).
Prolyl endopeptidase is a member of the prolyl oligopeptidase family of serine proteases. The isoelectric point of prolyl endopeptidase is 5.52 and its molecular weight is 80,769.59 Da, making it much larger than the classic serine proteases, trypsin (pdb ID: 1S81) and subtilisin (pdb ID: 1ST2) (1, 16). Oligopeptidase B (pdb ID: 2XE4), another member of the prolyl oligopeptidase family has only 25% primary structure similarity as shown by protein Blast (E=2e-51). But as with all the members of the prolyl oligopeptidase family, oligopeptidase B has the same overall tertiary structure as prolyl endopeptidase, as shown by DALI (Z= 46.1) (17). The enzymatic cleavage specificity of oligopeptidase B is different than that of prolyl endopeptidase; instead of cleaving at the endo side of prolyl residues, oligopeptidase B cleaves two adjacent basic residues and requires arginine for substrate binding (18). Oligopeptidase B, like prolyl endopeptidase, consists of an α/β hydrolase domain and a seven-bladed β propeller domain (19). The homology of oligopeptidase B and prolyl endopeptidase is higher in the catalytic domain, particularly around the active site, than in the propeller domain. Oligopeptidase B uses the same catalytic triad as prolyl endopeptidase, and in Leishmania major consists of Ser-577, Asp-662, and His-697 (19). Because all members of the prolyl oligopeptidase family contain the same catalytic triad, in order to prevent cross-reactivity of inhibitors, new drugs should target structural differences outside of the active site which are essential to catalysis (1).