László Polgár

Prolyl Oligopeptidase: An Unusual β-Propeller Domain Regulates Proteolysis

Abstract Prolyl oligopeptidase is a large cytosolic enzyme that belongs to a new class of serine peptidases. The enzyme is involved in the maturation and degradation of peptide hormones and neuropeptides, which relate to the induction of amnesia. The 1.4 A resolution crystal structure is presented here. The enzyme contains a peptidase domain with an α/β hydrolase fold, and its catalytic triad (Ser554, His680, Asp641) is covered by the central tunnel of an unusual β propeller. This domain makes prolyl oligopeptidase an oligopeptidase by excluding large structured peptides from the active site. In this way, the propeller protects larger peptides and proteins from proteolysis in the cytosol. The structure is also obtained with a transition state inhibitor, which may facilitate drug design to treat memory disorders.

The catalytic triad of serine peptidases.

Abstract.The catalytic action of serine peptidases depends on the interplay of a nucleophile, a general base and an acid. In the classic trypsin and subtilisin families this catalytic triad is composed of serine, histidine and aspartic acid residues and exhibits similar spatial arrangements, but the order of the residues in the amino acid sequence is different. By now several new families have been discovered, in which the nucleophile-base-acid pattern is generally conserved, but the individual components can vary. The variations illustrate how different groups and different protein structures achieve the same reaction.

Mercaptide-imidazolium ion-pair: The reactive nucleophile in papain catalysis

The hydrolysis of amino acid derivatives catalyzed by papain (EC 3.4.4.10) proceeds through the formation of an acyl-thiolenzyme intermediate (cf. refs. [ 1,2] ). The pH-rate profde of the formation of this intermediate displays a bell-shaped curve depending on the ionization of two groups with pK, values of about 4 and 8 [ 1,2]. In the light of the steric structure of the active site of papain [3,4] these pK, values can be assigned to His159 and Cys-25, respectively [ 5-71, although the pKa of 4 has earlier been attributed to the dissociation of a carboxyl group (cf. refs. [ 1,2] ). Despite the extensive studies on the mechanism of action, the exact catalytic role of Cys25 has not yet been established. It is widely accepted in the literature that during acyl-enzyme formation the thiol group reacts in its non-dissociated form assisted by general base catalysis [2,7-131. On the other hand, we have recently found [6] that alkylation of papain by ~oacetamides displays double sigmoid pHrate profdes with similar pKa values (4.0 and 8.4) as found in acylation reactions. Since Dz 0 effects could not be observed in these reactions [6], the double sigmoid curves were interpreted to indicate the existence of a mercaptide-imidazolium ion-pair in the catalytically active papain rather than in terms of a general base-catalyzed attack of the non-dissociated thiol group on the substrate. Starting from low pH values the pK, of 4.0 is characteristic of the formation of the ion-pair, whereas the pK, of 8.4 reflects decomposition of the ion-pair to free mercaptide ion and imidazole. The aim of this work was to test direcfly the presence of mercaptide ion in the catalytically active

Catalysis of serine oligopeptidases is controlled by a gating filter mechanism

Proteases have a variety of strategies for selecting substrates in order to prevent uncontrolled protein degradation. A recent crystal structure determination of prolyl oligopeptidase has suggested a way for substrate selection involving an unclosed seven‐bladed β‐propeller domain. We have engineered a disulfide bond between the first and seventh blades of the propeller, which resulted in the loss of enzymatic activity. These results provided direct evidence for a novel strategy of regulation in which oscillating propeller blades act as a gating filter during catalysis, letting small peptide substrates into the active site while excluding large proteins to prevent accidental proteolysis.

Structure, Function and Biological Relevance of Prolyl Oligopeptidase

A group of serine peptidases, the prolyl oligopeptidase family, cannot hydrolyze proteins and peptides containing more than 30 residues. The crystal structure of prolyl oligopeptidase (POP) has shown that the enzyme is composed of a peptidase domain with an alpha/beta hydrolase fold and a seven-bladed beta-propeller domain. This domain covers the catalytic triad and excludes large, structured peptides from the active site. The mechanism of substrate selection has been reviewed, along with the binding mode of the substrate and the catalytic mechanism, which differ from that of the classical serine peptidases in several features. POP is essentially a cytosolic enzyme and has been shown to be involved in a number of biological processes, but its precise function is still unknown. Many reports addressed experimentally the possible role of POP in cognitive and psychiatric processes, its involvement in the inositol phosphate signaling pathway, and its ability to metabolize bioactive peptides. Inhibitors were designed to reveal the cellular functions of POP and to treat neurological disorders. Other studies concerned the cellular localization of POP, its presumed interaction with the cytoskeletal elements, and its involvement in peptide/protein transport/secretion processes. The possible role of POP in Alzheimer disease is an intriguing issue, which is still debated. Recently, recombinant bacterial POPs have been investigated as potential therapeutics for celiac sprue, an autoimmune disease of small intestine caused by the intake of gluten proteins.

How can the mood stabilizer VPA limit both mania and depression

The mood stabilizing drugs commonly used to treat bipolar disorder--lithium, valproic acid (VPA), and carbamazepine (CBZ)--limit the frequency of swings to either manic or depressive states. We previously showed that these drugs all have a common action on cultured neurons, which can be reversed by the addition of either inositol or specific inhibitors of the enzyme prolyl oligopeptidase (PO). Inhibition of PO activity is reported to enhance phosphoinositide (PIns) signaling consistent with the suggestion that mood stabilizers inhibit PIns signaling. We now report that VPA directly inhibits recombinant PO activity, which would have the opposite effect on PIns signaling. This unexpected result suggests a model that could explain the dual action of VPA in stabilizing mood: we propose that euthymic mood is dependent on stable PIns signaling and that VPA may limit mood swings to mania by decreasing PIns signaling, and that it may limit mood swings to depression by inhibiting PO and thus increasing PIns signaling.

Structural relationship between lipases and peptidases of the prolyl oligopeptidase family

In prolyl oligopeptidase and its homologues, which constitute a new serine protease family, the order of the catalytic Ser and His residues in the amino acid sequence is the reverse of what is found in the trypsin and subtilisin families. The exact position of the third member of the catalytic triad, an Asp residue, has not yet been identified in the new family. Recent determination of the three‐dimensional structures of pancreatic and microbial lipases has shown that the order of their catalytic residues is Ser, Asp, His, and this fits the order Ser, His of prolyl oligopeptidase. However, there is no sequence homology between lipases and peptidases, except for a 10‐residue segment, which encompasses the essential Ser, and for the immediate vicinity of the catalytic Asp and His residues. This comparison identifies the catalytic Asp residue in the prolyl oligopeptidase family. The relative positions of the three catalytic residues in peptidases and microbial lipases were the same and this indicated structural and possibly evolutionary relationship between the two families.

Spectrophotometric determination of mercaptide ion, an activated form of SH-group in thiol enzymes

The dissociated form of a thiol group, the mercaptide ion, is a far better nucleophile than the nondissociated species [ 1 ]. Therefore, the thiol group of a simple SH-compound becomes tess reactive as its dissociation is reversed with decreasing pH. On the other hand, in thiol enzymes the reactivity of the essential thiol group often does not parallel the expected dissociation and it may be remarkably high even below the pK a of thiol group. A possible explanation for this high reactivity is offered by the studies on thiolsubtilisin [2] and papain [3], which indicate that the thiol group of these enzymes exists in the mercaptide ion form even at low pH due to an interaction with a neighboring histidine residue. In this paper we present a direct method to identify the dissociated form of the SH-group of thiol enzymes. The method is based on measuring the disappearance of the absorption band o f the mercaptide ion during alkylation. By means of this method in thiolsubtilisin the mercaptide ion could be detected in the pH-range where the mercaptide-imidazolium ion-pair has previously been assumed to exist [2].

Flexibility of prolyl oligopeptidase : Molecular dynamics and molecular framework analysis of the potential substrate pathways

The flexibility of prolyl oligopeptidase has been investigated using molecular dynamics (MD) and molecular framework approaches to delineate the route of the substrate to the active site. The selectivity of the enzyme is mediated by a seven‐bladed β‐propeller that in the crystal structure does not indicate the possible passage for the substrate to the catalytic center. Its open topology however, could allow the blades to move apart and let the substrate into the large central cavity. Flexibility analysis of prolyl oligopeptidase structure using the FIRST (Floppy Inclusion and Rigid Substructure Topology) approach and the atomic fluctuations derived from MD simulations demonstrated the rigidity of the propeller domain, which does not permit the substrate to approach the active site through this domain. Instead, a smaller tunnel at the inter‐domain region comprising the highly flexible N‐terminal segment of the peptidase domain and a facing hydrophilic loop from the propeller (residues 192–205) was identified by cross‐correlation analysis and essential dynamics as the only potential pathway for the substrate. The functional importance of the flexible loop has been also verified by kinetic analysis of the enzyme with a split loop. Catalytic effect of engineered disulfide bridges was rationalized by characterizing the concerted motions of the two domains. Proteins 2005.

Conformational Stability and Catalytic Activity of HIV-1 Protease Are Both Enhanced at High Salt Concentration*

The activity of human immunodeficiency virus protease is markedly increased at elevated salt concentration. The structural basis of this effect has been explored by several independent methods by using both the wild-type enzyme and its triple mutant (Q7K/L33I/L63I) (Mildner, A. M., Rothrock, D. J., Leone, J. W., Bannow, C. A., Lull, J. M., Reardon, I. M., Sarcich, J. L., Howe, W. J., Tomich, C.-S. C., Smith, C. W., Heinrikson, R. L., and Tomasselli, A. G.(1994) Biochemistry 33, 9405-9413), designed to better resist autolysis. Monitoring the intrinsic fluorescence of the two enzymes during urea-mediated denaturation has shown that at high NaCl concentration, both the conformational stability (ΔG0) and the transition midpoint (D) between the folded and unfolded states increase, indicating that the salt stabilizes the enzyme structure. These equilibrium data are supported by kinetic studies on the urea-mediated unfolding by measuring fluorescence change, red shifting in the maximum of the emission spectrum, and far- and near-UV CD. The salt effects observed in urea-mediated unfolding reactions prevail upon heat denaturation. All these findings support the existence of a two-state equilibrium between the folded and unfolded proteins. The pH dependence of fluorescence intensity indicated that the conformation of human immunodeficiency virus type 1 protease should change in the catalytically competent pH region. It is concluded that preferential hydration stabilizes the protease structure in the presence of salt, providing entropic contribution to enhance the catalytic activity.