Juliano L

References (3)

Title : The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity - Szeltner_2012_Biochim.Biophys.Acta_1834_98
Author(s) : Szeltner Z , Juhasz T , Szamosi I , Rea D , Fulop V , Modos K , Juliano L , Polgar L
Ref : Biochimica & Biophysica Acta , 1834 :98 , 2012
Abstract : Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189-209) and loop B (res. 577-608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.
ESTHER : Szeltner_2012_Biochim.Biophys.Acta_1834_98
PubMedSearch : Szeltner_2012_Biochim.Biophys.Acta_1834_98
PubMedID: 22940581
Gene_locus related to this paper: pig-ppce

Title : Fluorescence resonance energy transfer (FRET) peptides and cycloretro-inverso peptides derived from bradykinin as substrates and inhibitors of prolyl oligopeptidase - Gorrao_2007_Peptides_28_2146
Author(s) : Gorrao SS , Hemerly JP , Lima AR , Melo RL , Szeltner Z , Polgar L , Juliano MA , Juliano L
Ref : Peptides , 28 :2146 , 2007
Abstract : Prolyl oligopeptidase (POP, EC is a member of a family of serine peptidases with post-proline cleaving activity towards peptides. It is located in the cytosol in active form but without hydrolytic activity on proteins or peptides higher than 30 amino acids. Its function is not well defined, but it is involved in central nervous system disorders. Here, we studied the substrate specificity of wild type POP (POPwt) and its C255T variant lacking the non-catalytic Cys(255). This residue is located in the seven-bladed beta-propeller domain that regulates the activity of POP. Fluorescence resonance energy transfer (FRET) peptides were used with sequences derived from bradykinin-containing region of human kininogen and flanked by Abz (ortho-aminobenzoic acid) and EDDnp [N-ethylenediamine-(2,4-dinitrophenyl)]. The peptide Abz-GFSPFRQ-EDDnp was taken as leader substrate for the synthesis of five series of peptides modified at the P(3), P(2), P'(1), P'(2) and P'(3) residues. The optimal amino acids in each position for POPwt resulted in the sequence RRPYIR that is very similar to the C-terminal sequence of neurotensin. The cyclic peptides c(G((n))FSPFR) (n=1-4) were hydrolyzed by POP; their cycloretro and cycloretro-inverso analogues were inhibitors in the micromolar range. The differences between POPwt and its C255T mutant in the hydrolysis of the series derived from Abz-GFSPFRQ-EDDnp were restricted to the non-prime site of the substrates. The kinetic data of hydrolysis and inhibition by the cyclic peptides are consistent with the structures of POP-substrate/inhibitor complexes and with the substrate specificity data obtained with linear FRET peptides. All together, these results give information about the POP-substrate/inhibitor interactions that further complete knowledge of this important oligopeptidase.
ESTHER : Gorrao_2007_Peptides_28_2146
PubMedSearch : Gorrao_2007_Peptides_28_2146
PubMedID: 17904692

Title : Electrostatic environment at the active site of prolyl oligopeptidase is highly influential during substrate binding - Szeltner_2003_J.Biol.Chem_278_48786
Author(s) : Szeltner Z , Rea D , Renner V , Juliano L , Fulop V , Polgar L
Ref : Journal of Biological Chemistry , 278 :48786 , 2003
Abstract : The positive electrostatic environment of the active site of prolyl oligopeptidase was investigated by using substrates with glutamic acid at positions P2, P3, P4, and P5, respectively. The different substrates gave various pH rate profiles. The pKa values extracted from the curves are apparent parameters, presumably affected by the nearby charged residues, and do not reflect the ionization of a simple catalytic histidine as found in the classic serine peptidases like chymotrypsin and subtilisin. The temperature dependence of kcat/Km did not produce linear Arrhenius plots, indicating different changes in the individual rate constants with the increase in temperature. This rendered it possible to calculate these constants, i.e. the formation (k1) and decomposition (k-1) of the enzyme-substrate complex and the acylation constant (k2), as well as the corresponding activation energies. The results have revealed the relationship between the complex Michaelis parameters and the individual rate constants. Structure determination of the enzyme-substrate complexes has shown that the different substrates display a uniform binding mode. None of the glutamic acids interacts with a charged group. We conclude that the specific rate constant is controlled by k1 rather than k2 and that the charged residues from the substrate and the enzyme can markedly affect the formation but not the structure of the enzyme-substrate complexes.
ESTHER : Szeltner_2003_J.Biol.Chem_278_48786
PubMedSearch : Szeltner_2003_J.Biol.Chem_278_48786
PubMedID: 14514675
Gene_locus related to this paper: pig-ppce