Proteases have a variety of strategies for selecting substrates in order to prevent uncontrolled protein degradation. In prolyl oligopeptidase, the narrow entrance of the propeller opposite the active site is much smaller than the diameter of an average peptide, so that the substrate cannot enter through it. We have demonstrated that the propeller has stable structure, indicating that it cannot open up and the substrate should approach the catalytic site through a tunnel between the peptidase and the propeller domains. This requires the concerted movements of the two domains. This mechanism was corroborated by X-ray crystallography.

We have determined the crystal structures and kinetic parameters of various active site mutants complexed with peptides/inhibitors. These studies indicated that substrate binding is restricted to the P4-P2’ region [1] and the contributions of Asp641 were very much dependent on the substrate-leaving group [2], which was not the case for the classic serine peptidases. We have also delineated the catalytic role of the oxyanion binding site using the Tyr473Phe enzyme variant [3]. Interestingly, the propeller domain not just excludes large peptides from the active site but also contributes to the catalysis [4].

We are going to investigate the biological functions of POP. Specifically, we examine the possible interactions suggested between POP with the microtubular system and between POP and GAP43, a protein that expresses at the neuronal growth cone. Our other aim is to isolate the real physiological substrates of POP by the aid of an engineered enzyme variant that may trap the substrate, and to characterize the pathways in which the substrates are involved. A further objective is to find the regulator peptide(s) of the inositol cycle in which the POP is implicated.


Oligopeptidase B

The enzyme is found in Gram-negative bacteria and trypanosomes and is involved in host cell invasion, thus being an important target for drug design. We have shown that oligopeptidase B is specific for substrates with a pair of basic residues at positions P1 and P2 [5]. On the basis of a three-dimensional model, two carboxyl dyads (Asp460 and Asp462 and Glu576 and Glu578) were assigned as binding sites for arginines P1 and P2, respectively. The dyads were shown to be involved in several events: (i) substrate binding, (ii) substrate inhibition at high substrate concentrations, and (iii) enzyme activation at millimolar CaCl2 concentrations with substrates having one arginine [6]. The OH group of Tyr452 is part of the oxyanion binding site, which stabilizes the transition state of the reaction. Using the Tyr452Phe variant, we have shown that the catalytic contribution of the OH group depends on the substrate and that the catalysis is, unusually, an entropy-driven process [6].

Acylaminoacyl peptidase

In contrast to the monomer prolyl oligopeptidase and oligopeptidase B, acylaminoacyl peptidase is an oligomer enzyme, and the significance of assembly of subunits is of primary interest. We have studied the mesophilic tetramer enzyme from liver and the thermophilic dimer acylaminoacl peptidase from archaeon Aeropyrum pernix K1. We isolated the enzyme from porcine liver, and cloned it from human liver (91% sequence identity). The study of the His507Ala variant indicated that the oxyanion binding site of acylaminoacyl peptidase is different from that of the other enzymes of the family [7]. The crystal structure of the thermophilic enzyme was determined [8] (Figure 2), which rendered it possible to investigate the catalytic mechanism and the importance of oligomerization of the enzyme, using site specific mutagenesis. We have found that the thermophilic enzyme thought to be an exopeptidase also displays endopeptidase activity [9]. Preliminary results with the His367Ala variant, which correspond to the His507Ala variant of the mammalian enzyme, shows that the main chain of Gly369, which stabilizes the oxyanion of the catalytic intermediate, moves away from its native position, thereby eliciting 2-3 orders of magnitude reduction in the specificity rate constant. Site specific modifications at the interface of the dimer structure allow us to prepare the monomer form and study its kinetic and structural properties. Structural studies of another thermophilic acylaminoacyl peptidase indicate a higher degree of oligomerization.

 

References:

1. Fülöp V, Szeltner Z, Renner V and Polgár L (2001)
   Structures of prolyl oligopeptidase substrate/inhibitor complexes. Use of inhibitor binding for titration of the catalytic histidine residue.
   J Biol Chem 276, 1262-6 [PubMed]
2. Szeltner Z, Rea D, Juhász T, Renner V, Mucsi Z, Orosz G, Fülöp V and Polgár L (2002)
   Substrate-dependent competency of the catalytic triad of prolyl oligopeptidase.
   J Biol Chem 277, 44597-605 [PubMed]
3. Szeltner Z, Renner V and Polgár L (2000)
   Substrate- and pH-dependent contribution of oxyanion binding site to the catalysis of prolyl oligopeptidase, a paradigm of the serine oligopeptidase family.
   Protein Sci 9, 353-60 [PubMed]
4. Szeltner Z, Renner V and Polgár L (2000)
   The noncatalytic beta-propeller domain of prolyl oligopeptidase enhances the catalytic capability of the peptidase domain.
   J Biol Chem 275, 15000-5 [PubMed]
5. Polgár L (1997)
   A potential processing enzyme in prokaryotes: oligopeptidase B, a new type of serine peptidase.
   Proteins 28, 375-9 [PubMed]
6. Juhász T, Szeltner Z, Renner V and Polgár L (2002)
   Role of the oxyanion binding site and subsites S1 and S2 in the catalysis of oligopeptidase B, a novel target for antimicrobial chemotherapy.
   Biochemistry-us 41, 4096-106 [PubMed]
7. Kiss AL, Szeltner Z, Fülöp V and Polgár L (2004)
   His507 of acylaminoacyl peptidase stabilizes the active site conformation, not the catalytic intermediate.
   FEBS Lett 571, 17-20 [PubMed]
8. Bartlam M, Wang G, Yang H, Gao R, Zhao X, Xie G, Cao S, Feng Y and Rao Z (2004)
   Crystal structure of an acylpeptide hydrolase/esterase from Aeropyrum pernix K1.
   Structure 12, 1481-8 [PubMed]
9. Kiss AL, Hornung B, Rádi K, Gengeliczki Z, Sztáray B, Juhász T, Szeltner Z, Harmat V and Polgár L (2007)
   The acylaminoacyl peptidase from Aeropyrum pernix K1 thought to be an exopeptidase displays endopeptidase activity.
   J Mol Biol 368, 509-20 [PubMed]