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Acylaminoacyl-Peptidase
The Experts below are selected from a list of 102 Experts worldwide ranked by ideXlab platform
László Polgár – 1st expert on this subject based on the ideXlab platform
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Hipertermofil szerin oligopeptidázok szerkezete és működése = Structure and function of hyperthermophilic serine oligopeptidases
, 2020Co-Authors: Gábor Náray-szabó, Andras Kiss, Veronika Harmat, Tünde Juhász, Klarissza Domokos, Andrea Bodor, Ilona Hudáky, András Láng, Gábor Pohl, László PolgárAbstract:A kutatas fő celja az egyikunk altal felfedezett es jellemzett prolil oligopeptidaz csaladhoz tartozo enzimek (prolil oligopeptidaz, acilaminoacil peptidaz) műkodesi mechanizmusanak reszletes tisztazasa es kulonboző ligandumokkal kepezett komplexeinek szerkezeti elemzese volt. Az enzimek kulonboző variansainak műkodeset kombinalt modszerekkel, oldatkinetikai es feherjekrisztallografiai vizsgalatokkal, valamint molekulamodellezessel jellemeztuk. Megallapitottuk, hogy ahhoz, hogy a katalitikus hely aktiv konformacioba billenjen, nem elegendőek egy kismeretű szubsztrattal kialakitott nem-kovalens komplex kolcsonhatasai, hanem az enzim becsukodasa es interdomen kolcsonhatasainak atrendeződese szukseges – ez biztositja, hogy nagyobb polipeptideket es feherjeket az enzim nem hidrolizal el. Analogiat talaltunk tobb mas feherje (kalmodulin, dUTPaz es MASP-1), valamint az acilaminoacil peptidaz ligandumkotesi mechanizmusa kozott. A kotőhely nyitottsaganak merteke, es becsukodasi kepessege osszefuggesbe hozhato a szelesebb szelektivitassal (kalmodulin es MASP-1). Az aktiv hellyel kozvetlenul nem erintkező konzervalt aminosavak tavol hato, konzervalt hidrogenhid-halozatok reven vesznek reszt a szubsztrat stabilizalasaban (dUTPaz). A PCM modellel kiegeszitett kvantumkemiai modszerrel vizsgaltuk a feherje gerinc proton es szen NMR kemiai eltolodasait a konformacio fuggvenyeben, es megallapitottuk, hogy a gazfazisu modellhez kepest javult az egyezes a kiserleti adatokkal. | The main goal of our studies was to clarify the detailed mechanism of action of enzymes, belonging to the prolyl oligopeptidase family, discovered by one of us, as well as structural analysis of their complexes with various ligands. Activity of enzyme variants was characterised by combined methods, like solution kinetics, protein crystallography and molecular modelling. We found that, in order to switch the active site into an active conformation, it is not enough to establish non-covalent interactions with small substrates, rather closing of the enzyme and reorganisation of the interdomain interactions is needed. This ensures that larger polypeptides and proteins are not hydrolysed by the enzyme. We found an analogy among mechanisms of action of other proteins (calmodulin, dUTPase and MASP-1) and acylaminoacyl peptidase. The extent of opening at the binding site and ability to close can be related to broader selectivity (calmodulin and MASP-1). Conserved amino acid residues, not directly connected to the active site, participate in substrate stabilisation via extended, conserved H-bond networks (dUTPase). We investigated NMR proton and carbon chemical shifts of the protein backbone as a function of the conformation with a quantum chemical method extended by the PCM model and found that that the agreement with experimental data improved as compared to the gas-phase model.
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Oigopeptidázok regulációs és katalitikus mechanizmusa = Regulatory and catalytic mechanisms of oligopeptidases
, 2020Co-Authors: László Polgár, Andras Kiss, Tünde Juhász, Veronika Renner, Zoltán SzeltnerAbstract:Az oligopeptidazok csak kisebb peptideket hidrolizalnak. Ennek okat a proliloligopeptidaz (POP) enzimnel vizsgaltuk, amelynek fontos szerepe van a kozponti idegrendszer műkodeseben. Az altalunk meghatarozott kristalyszerkezet azt mutatta, hogy az enzim egy peptidaz es egy propeller domenből all, es az utobbi gatolja a nagyobb szubsztratoknak az aktiv centrumhoz valo jutasat. Diszulfid keresztkotesekkel es stabilitas vizsgalatokkal kimutattuk, hogy a zart propeller nem engedheti be a szubsztratot, az csak a ket domen kozott, azok flexibilitasa reven juthat az aktiv centrumhoz. Az itt megnyilo kapu azonban kiszűri a feherjeket. Kimutattuk tovabba, hogy a valproinsav, a depresszio egyik gyogyszere, gatolja az enzim műkodeset. Ugyancsak megallapitottuk, hogy a POP csaladba tartozo PREPL feherje, melynek hianya sulyos betegseget okoz, nem rendelkezik hidrolitikus aktivitassal. Egy masik, szinten a POP csaladba tartozo enzimről, az acilaminoacil peptidazrol kimutattuk, hogy a katalizisben resztvevő oxianion kotőhely műkodeset jelentősen karositja egy a kotőhelyen kivuli aminosav mutacioja. Rontgen krisztallografiaval igazoltuk, hogy a mutacio megvaltoztatja a kotőhely szerkezetet. Lenyeges kulonbseget talaltunk az emlős es egy bakterialis acilaminoacil peptidaz kozott. Mig az emlős enzim valodi exopeptidaz, acilaminosavat hasit le a peptidlanc vegeről, addig a bakterialis enzim endopeptidaz aktivitassal is rendelkezik. Ez arra mutat, hogy az enzim a fejlődes soran specializalodott. | Oligopeptidases hydrolyze small peptides only. The reason of the limitation was studied using prolyloligopeptidase (POP), which is involved in the function of the central nervous system. As we have shown the enzyme is composed of a peptidase and a propeller domain, the latter preventing the access of the substrate to the active site. Using disulfide cross-linking, molecular dynamics calculations and stability investigations, we have demonstrated that the substrate enters the active site between the domains, thanks to the flexibility of the protein. This gate, however, excludes the protein from the catalytic centre. We have also demonstrated that valproic acid, a drug for treating bipolar depression, inhibits POP. Furthermore, the PREPL protein of the POP family, the lack of which causes serious illness, was shown to be inactive. Acylaminoacyl peptidase, an important member of POP family, has also been investigated. A mutation outside the oxyanion binding site considerably impaired the catalytic activity due to a distortion in the structure of the oxyanion binding site, as demonstrated by X-ray crystallography. A significant difference was observed between the mammalian and a bacterial acylaminoacyl peptidase. While the mammalian enzyme proved to be a true exopeptidase cleaving acylaminoacid from the N-terminus of peptides, the bacterial enzyme also displayed endopeptidase activity, indicating that the enzyme specialized during evolution.
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Structure and catalysis of acylaminoacyl peptidase: closed and open subunits of a dimer oligopeptidase.
Journal of Biological Chemistry, 2010Co-Authors: Veronika Harmat, Zoltán Szeltner, Anna Palló, Gábor Náray-szabó, K. Domokos, Dóra K. Menyhárd, I. Szamosi, Tamás Beke-somfai, László PolgárAbstract:Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.
Zoltán Szeltner – 2nd expert on this subject based on the ideXlab platform
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Oigopeptidázok regulációs és katalitikus mechanizmusa = Regulatory and catalytic mechanisms of oligopeptidases
, 2020Co-Authors: László Polgár, Andras Kiss, Tünde Juhász, Veronika Renner, Zoltán SzeltnerAbstract:Az oligopeptidazok csak kisebb peptideket hidrolizalnak. Ennek okat a proliloligopeptidaz (POP) enzimnel vizsgaltuk, amelynek fontos szerepe van a kozponti idegrendszer műkodeseben. Az altalunk meghatarozott kristalyszerkezet azt mutatta, hogy az enzim egy peptidaz es egy propeller domenből all, es az utobbi gatolja a nagyobb szubsztratoknak az aktiv centrumhoz valo jutasat. Diszulfid keresztkotesekkel es stabilitas vizsgalatokkal kimutattuk, hogy a zart propeller nem engedheti be a szubsztratot, az csak a ket domen kozott, azok flexibilitasa reven juthat az aktiv centrumhoz. Az itt megnyilo kapu azonban kiszűri a feherjeket. Kimutattuk tovabba, hogy a valproinsav, a depresszio egyik gyogyszere, gatolja az enzim műkodeset. Ugyancsak megallapitottuk, hogy a POP csaladba tartozo PREPL feherje, melynek hianya sulyos betegseget okoz, nem rendelkezik hidrolitikus aktivitassal. Egy masik, szinten a POP csaladba tartozo enzimről, az acilaminoacil peptidazrol kimutattuk, hogy a katalizisben resztvevő oxianion kotőhely műkodeset jelentősen karositja egy a kotőhelyen kivuli aminosav mutacioja. Rontgen krisztallografiaval igazoltuk, hogy a mutacio megvaltoztatja a kotőhely szerkezetet. Lenyeges kulonbseget talaltunk az emlős es egy bakterialis acilaminoacil peptidaz kozott. Mig az emlős enzim valodi exopeptidaz, acilaminosavat hasit le a peptidlanc vegeről, addig a bakterialis enzim endopeptidaz aktivitassal is rendelkezik. Ez arra mutat, hogy az enzim a fejlődes soran specializalodott. | Oligopeptidases hydrolyze small peptides only. The reason of the limitation was studied using prolyloligopeptidase (POP), which is involved in the function of the central nervous system. As we have shown the enzyme is composed of a peptidase and a propeller domain, the latter preventing the access of the substrate to the active site. Using disulfide cross-linking, molecular dynamics calculations and stability investigations, we have demonstrated that the substrate enters the active site between the domains, thanks to the flexibility of the protein. This gate, however, excludes the protein from the catalytic centre. We have also demonstrated that valproic acid, a drug for treating bipolar depression, inhibits POP. Furthermore, the PREPL protein of the POP family, the lack of which causes serious illness, was shown to be inactive. Acylaminoacyl peptidase, an important member of POP family, has also been investigated. A mutation outside the oxyanion binding site considerably impaired the catalytic activity due to a distortion in the structure of the oxyanion binding site, as demonstrated by X-ray crystallography. A significant difference was observed between the mammalian and a bacterial acylaminoacyl peptidase. While the mammalian enzyme proved to be a true exopeptidase cleaving acylaminoacid from the N-terminus of peptides, the bacterial enzyme also displayed endopeptidase activity, indicating that the enzyme specialized during evolution.
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Catalytically distinct states captured in a crystal lattice: the substrate-bound and scavenger states of acylaminoacyl peptidase and their implications for functionality.
Acta Crystallographica Section D-biological Crystallography, 2015Co-Authors: Dóra K. Menyhárd, Zoltán Szeltner, I. Szamosi, Zoltán Orgován, Veronika HarmatAbstract:: Acylaminoacyl peptidase (AAP) is an oligopeptidase that only cleaves short peptides or protein segments. In the case of AAP from Aeropyrum pernix (ApAAP), previous studies have led to a model in which the clamshell-like opening and closing of the enzyme provides the means of substrate-size selection. The closed form of the enzyme is catalytically active, while opening deactivates the catalytic triad. The crystallographic results presented here show that the open form of ApAAP is indeed functionally disabled. The obtained crystal structures also reveal that the closed form is penetrable to small ligands: inhibitor added to the pre-formed crystal was able to reach the active site of the rigidified protein, which is only possible through the narrow channel of the propeller domain. Molecular-dynamics simulations investigating the structure of the complexes formed with longer peptide substrates showed that their binding within the large crevice of the closed form of ApAAP leaves the enzyme structure unperturbed; however, their accessing the binding site seems more probable when assisted by opening of the enzyme. Thus, the open form of ApAAP corresponds to a scavenger of possible substrates, the actual cleavage of which only takes place if the enzyme is able to re-close.
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Structure and catalysis of acylaminoacyl peptidase: closed and open subunits of a dimer oligopeptidase.
Journal of Biological Chemistry, 2010Co-Authors: Veronika Harmat, Zoltán Szeltner, Anna Palló, Gábor Náray-szabó, K. Domokos, Dóra K. Menyhárd, I. Szamosi, Tamás Beke-somfai, László PolgárAbstract:Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.
Andras Kiss – 3rd expert on this subject based on the ideXlab platform
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Hipertermofil szerin oligopeptidázok szerkezete és működése = Structure and function of hyperthermophilic serine oligopeptidases
, 2020Co-Authors: Gábor Náray-szabó, Andras Kiss, Veronika Harmat, Tünde Juhász, Klarissza Domokos, Andrea Bodor, Ilona Hudáky, András Láng, Gábor Pohl, László PolgárAbstract:A kutatas fő celja az egyikunk altal felfedezett es jellemzett prolil oligopeptidaz csaladhoz tartozo enzimek (prolil oligopeptidaz, acilaminoacil peptidaz) műkodesi mechanizmusanak reszletes tisztazasa es kulonboző ligandumokkal kepezett komplexeinek szerkezeti elemzese volt. Az enzimek kulonboző variansainak műkodeset kombinalt modszerekkel, oldatkinetikai es feherjekrisztallografiai vizsgalatokkal, valamint molekulamodellezessel jellemeztuk. Megallapitottuk, hogy ahhoz, hogy a katalitikus hely aktiv konformacioba billenjen, nem elegendőek egy kismeretű szubsztrattal kialakitott nem-kovalens komplex kolcsonhatasai, hanem az enzim becsukodasa es interdomen kolcsonhatasainak atrendeződese szukseges – ez biztositja, hogy nagyobb polipeptideket es feherjeket az enzim nem hidrolizal el. Analogiat talaltunk tobb mas feherje (kalmodulin, dUTPaz es MASP-1), valamint az acilaminoacil peptidaz ligandumkotesi mechanizmusa kozott. A kotőhely nyitottsaganak merteke, es becsukodasi kepessege osszefuggesbe hozhato a szelesebb szelektivitassal (kalmodulin es MASP-1). Az aktiv hellyel kozvetlenul nem erintkező konzervalt aminosavak tavol hato, konzervalt hidrogenhid-halozatok reven vesznek reszt a szubsztrat stabilizalasaban (dUTPaz). A PCM modellel kiegeszitett kvantumkemiai modszerrel vizsgaltuk a feherje gerinc proton es szen NMR kemiai eltolodasait a konformacio fuggvenyeben, es megallapitottuk, hogy a gazfazisu modellhez kepest javult az egyezes a kiserleti adatokkal. | The main goal of our studies was to clarify the detailed mechanism of action of enzymes, belonging to the prolyl oligopeptidase family, discovered by one of us, as well as structural analysis of their complexes with various ligands. Activity of enzyme variants was characterised by combined methods, like solution kinetics, protein crystallography and molecular modelling. We found that, in order to switch the active site into an active conformation, it is not enough to establish non-covalent interactions with small substrates, rather closing of the enzyme and reorganisation of the interdomain interactions is needed. This ensures that larger polypeptides and proteins are not hydrolysed by the enzyme. We found an analogy among mechanisms of action of other proteins (calmodulin, dUTPase and MASP-1) and acylaminoacyl peptidase. The extent of opening at the binding site and ability to close can be related to broader selectivity (calmodulin and MASP-1). Conserved amino acid residues, not directly connected to the active site, participate in substrate stabilisation via extended, conserved H-bond networks (dUTPase). We investigated NMR proton and carbon chemical shifts of the protein backbone as a function of the conformation with a quantum chemical method extended by the PCM model and found that that the agreement with experimental data improved as compared to the gas-phase model.
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Oigopeptidázok regulációs és katalitikus mechanizmusa = Regulatory and catalytic mechanisms of oligopeptidases
, 2020Co-Authors: László Polgár, Andras Kiss, Tünde Juhász, Veronika Renner, Zoltán SzeltnerAbstract:Az oligopeptidazok csak kisebb peptideket hidrolizalnak. Ennek okat a proliloligopeptidaz (POP) enzimnel vizsgaltuk, amelynek fontos szerepe van a kozponti idegrendszer műkodeseben. Az altalunk meghatarozott kristalyszerkezet azt mutatta, hogy az enzim egy peptidaz es egy propeller domenből all, es az utobbi gatolja a nagyobb szubsztratoknak az aktiv centrumhoz valo jutasat. Diszulfid keresztkotesekkel es stabilitas vizsgalatokkal kimutattuk, hogy a zart propeller nem engedheti be a szubsztratot, az csak a ket domen kozott, azok flexibilitasa reven juthat az aktiv centrumhoz. Az itt megnyilo kapu azonban kiszűri a feherjeket. Kimutattuk tovabba, hogy a valproinsav, a depresszio egyik gyogyszere, gatolja az enzim műkodeset. Ugyancsak megallapitottuk, hogy a POP csaladba tartozo PREPL feherje, melynek hianya sulyos betegseget okoz, nem rendelkezik hidrolitikus aktivitassal. Egy masik, szinten a POP csaladba tartozo enzimről, az acilaminoacil peptidazrol kimutattuk, hogy a katalizisben resztvevő oxianion kotőhely műkodeset jelentősen karositja egy a kotőhelyen kivuli aminosav mutacioja. Rontgen krisztallografiaval igazoltuk, hogy a mutacio megvaltoztatja a kotőhely szerkezetet. Lenyeges kulonbseget talaltunk az emlős es egy bakterialis acilaminoacil peptidaz kozott. Mig az emlős enzim valodi exopeptidaz, acilaminosavat hasit le a peptidlanc vegeről, addig a bakterialis enzim endopeptidaz aktivitassal is rendelkezik. Ez arra mutat, hogy az enzim a fejlődes soran specializalodott. | Oligopeptidases hydrolyze small peptides only. The reason of the limitation was studied using prolyloligopeptidase (POP), which is involved in the function of the central nervous system. As we have shown the enzyme is composed of a peptidase and a propeller domain, the latter preventing the access of the substrate to the active site. Using disulfide cross-linking, molecular dynamics calculations and stability investigations, we have demonstrated that the substrate enters the active site between the domains, thanks to the flexibility of the protein. This gate, however, excludes the protein from the catalytic centre. We have also demonstrated that valproic acid, a drug for treating bipolar depression, inhibits POP. Furthermore, the PREPL protein of the POP family, the lack of which causes serious illness, was shown to be inactive. Acylaminoacyl peptidase, an important member of POP family, has also been investigated. A mutation outside the oxyanion binding site considerably impaired the catalytic activity due to a distortion in the structure of the oxyanion binding site, as demonstrated by X-ray crystallography. A significant difference was observed between the mammalian and a bacterial acylaminoacyl peptidase. While the mammalian enzyme proved to be a true exopeptidase cleaving acylaminoacid from the N-terminus of peptides, the bacterial enzyme also displayed endopeptidase activity, indicating that the enzyme specialized during evolution.
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Characterization of a novel acylaminoacyl peptidase with hexameric structure and endopeptidase activity.
Biochimica et Biophysica Acta, 2009Co-Authors: Zoltán Szeltner, Andras Kiss, Gábor Náray-szabó, Veronika Harmat, Klarissza Domokos, László PolgárAbstract:Abstract We have overexpressed in E. coli, purified and investigated the kinetic, thermodynamic and biophysical properties of an acylaminoacyl peptidase (AAP), from the thermophile Pyrococcus horikoshii (PhAAP). It was shown that the electrostatic environment of the catalytic site of PhAAP substantially influenced the pH dependence of the specificity rate constant (kcat / Km). However, 0.3 M NaCl, which depressed the electrostatic effects, simplified the complex pH-rate profile. The rate of formation of the enzyme–substrate complex (k1) was obtained from a non-linear Arrhenius plot. The lack of substrate leaving group effects indicated that k1 is the rate determining step in the catalysis. DSC and CD measurements demonstrated that PhAAP displayed a stable structure in the catalytically competent pH range. It was shown that PhAAP is not just an acylaminoacyl peptidase, but it also has an endopeptidase activity and so differs from the mammalian AAPs. Size exclusion chromatography with PhAAP revealed a hexameric structure, which is unique among the known members of the prolyl oligopeptidase family that includes AAPs and suggests that its cellular function may be different from that of the dimeric AAP also found in the same organism.