8 research outputs found

    Still a Long Way to Fully Understanding the Molecular Mechanism of Escherichia coli Purine Nucleoside Phosphorylase

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    The results of several decades of studying the catalytic mechanism of Escherichia colt purine nucleoside phosphorylases (PNP) by solution studies and crystal structure determinations are presented. Potentially PNPs can be used for enzyme-activating prodrug gene therapy against solid tumours because of the differences in specificity between human and E. coli PNPs. Biologically active form of PNP from E. coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. Two conformations of the active site are possible after substrate(s) binding: open and closed. A series of determined 3D-structures of PNP binary and ternary complexes facilitated the prediction of the main steps in the catalytic mechanism. For their validation the active site mutants: Arg24Ala, Asp204Ala, Arg217Ala, Asp204Asn and double mutant Asp204Ala/Arg217Ala were prepared, The activity tests confirm that catalysis involves protonation of the purine base at position N7 and give better insight into the cooperativity between subunits in this oligomeric enzyme

    Crystallographic snapshots of ligand binding to hexameric purine nucleoside phosphorylase and kinetic studies give insight into the mechanism of catalysis

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    Purine nucleoside phosphorylase (PNP) catalyses the cleavage of the glycosidic bond of purine nucleosides using phosphate instead of water as a second substrate. PNP from Escherichia coli is a homohexamer, build as a trimer of dimers, and each subunit can be in two conformations, open or closed. This conformational change is induced by the presence of phosphate substrate, and very likely a required step for the catalysis. Closing one active site strongly affects the others, by a yet unclear mechanism and order of events. Kinetic and ligand binding studies show strong negative cooperativity between subunits. Here, for the first time, we managed to monitor the sequence of nucleoside binding to individual subunits in the crystal structures of the wild-type enzyme, showing that first the closed sites, not the open ones, are occupied by the nucleoside. However, two mutations within the active site, Asp204Ala/Arg217Ala, are enough not only to significantly reduce the effectiveness of the enzyme, but also reverse the sequence of the nucleoside binding. In the mutant the open sites, neighbours in a dimer of those in the closed conformation, are occupied as first. This demonstrates how important for the effective catalysis of Escherichia coli PNP is proper subunit cooperation

    Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent

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    E. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein鈥搇igand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule

    Biophysical studies of purine nucleoside phosphorylase from E. coli - mechanism of catalysis, relationship between protein function and its oligomeric structure

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    Fosforylaza nukleozyd贸w purynowych (PNP) z Escherichia coli katalizuje odwracaln膮 reakcj臋 fosforolitycznego rozszczepienia nukleozyd贸w purynowych na odpowiedni膮 zasad臋 purynow膮 i pentozo-1-fosforan. Bia艂ko jest homoheksamerem i w formie apo struktura wszystkich podjednostek jest taka sama. Dane literaturowe, dost臋pne w momencie rozpoczynania tej pracy, sugerowa艂y, 偶e strukturalnie bia艂ko jest trimerem dimer贸w, poniewa偶 miejsce aktywne ka偶dej podjednostki w dimerze jest wsp贸艂tworzone przez dwa aminokwasy s膮siada z tego samego dimeru. To sugerowa艂o r贸wnie偶, 偶e podstawow膮 jednostk膮 katalityczn膮 bia艂ka jest dimer. Wyniki bada艅 w roztworze, wydawa艂y si臋 potwierdza膰 t臋 hipotez臋, poniewa偶, zauwa偶ono niemichaelisowsk膮 kinetyk臋 PNP wobec jon贸w fosforanowych ze wsp贸艂czynnikiem Hilla r贸wnym 0,5, wskazuj膮cym na siln膮 negatywn膮 kooperacj臋 [1]. Kilka, dost臋pnych w literaturze struktur krystalograficznych apo-enzymu i r贸偶nych kompleks贸w nie wyja艣nia艂o mechanizmu, za pomoc膮 kt贸rego informacja jest przekazywana mi臋dzy podjednostkami lub miejscami aktywnymi enzymu, ani te偶 jaka jest rola kooperacji podjednostek w katalizie. W 2002 roku, zosta艂a uzyskana struktura krystalograficzna, w kt贸rej zaobserwowano zamkni臋t膮 konformacj臋 cz臋艣ci miejsc aktywnych, po jednym w ka偶dym dimerze, pozosta艂e podjednostki zachowa艂y konformacj臋 otwart膮 miejsca aktywnego, jak w formie apo. Na podstawie tej struktury, zosta艂 zaproponowany mechanizm katalizy, opisuj膮cy sekwencj臋 zdarze艅 w miejscu aktywnym [2], kt贸ra polega艂aby na zamkni臋ciu miejsca aktywnego przy udziale Arg24. W tej konformacji miejsca aktywnego reszta Asp204, przy wsp贸艂pracy Arg217, mo偶e uprotonowa膰 nukleozyd, co zmienia rozk艂ad 艂adunku na pier艣cieniu purynowym, prowadzi do destabilizacji i p臋kni臋cia wi膮zania glikozydowego. Celem pracy by艂a weryfikacja zaproponowanego mechanizmu katalizy, poprzez badania mutant贸w miejsca aktywnego, oraz wyja艣nienie mechanizmu i znaczenia kooperacji mi臋dzy podjednostkami heksamerycznego bia艂ka. Pierwsz膮 przes艂ank膮, 偶e kooperacja zachodzi nie tylko wewn膮trz dimeru, ale r贸wnie偶 pomi臋dzy nimi, by艂y krzywe wi膮zania jon贸w fosforanowych, dla opisu kt贸rych model wi膮zania z dwiema sta艂ymi dysocjacji okaza艂 si臋 niewystarczaj膮cy. Wi膮zanie opisywane trzema sta艂ymi dysocjacji zosta艂o potwierdzone przy pomocy kilku technik eksperymentalnych. Zwi膮zanie pierwszego jonu fosforanowego prowadzi do powstania w bia艂ku trzech rodzaj贸w miejsc aktywnych, jednego w konformacji zamkni臋tej i dw贸ch w konformacji otwartej, charakteryzuj膮cych si臋 r贸偶nym powinowactwem do ligandu. Wobec tego fakt, 偶e wi膮zanie nukleozydu okaza艂o si臋 by膰 opisywane r贸wnie偶 trzema sta艂ymi nie by艂 zaskoczeniem. Istnienie trzech rodzaj贸w miejsc wi膮偶膮cych zaobserwowano tak偶e w strukturach krystalograficznych. Poprzez destabilizacj臋 kontakt贸w pomi臋dzy dimerami w heksamerze, uzyskano dimery, kt贸re okaza艂y si臋 nie by膰 stabilne na tyle, aby prowadzi膰 efektywn膮 kataliz臋. Mutuj膮c Asp204, Arg217 i Arg24 na alanin臋 i prowadz膮c badania kinetyczne wzgl臋dem naturalnych, a tak偶e imituj膮cych stan uprotonowany, substrat贸w, wykazano, 偶e, jak zaproponowano w 2002 roku, zmiana konformacyjna i protonacja nukleozydu s膮 niezb臋dnymi etapami katalizy. Na podstawie przeprowadzonych bada艅 zaproponowano r贸wnie偶 mechanizm katalizy, uwzgl臋dniaj膮cy kooperacj臋 pomi臋dzy podjednostkami bia艂ka w dimerze. W warunkach nasycenia substratami podjednostki otwarte i zamkni臋te w dimerach prowadz膮 kataliz臋 w 艣cis艂ej wsp贸艂pracy. Gdy w zamkni臋tej podjednostce zachodzi reakcja fosforolizy, podjednostka otwarta w艂a艣nie j膮 zako艅czy艂a, nast臋puje uwolnienie produkt贸w i zwi膮zanie kolejnej pary substrat贸w, zamkni臋cie tej podjednostki i cykl zaczyna si臋 od nowa. Jest to mechanizm zwany w literaturze angloj臋zycznej flip-flop. Poza istnieniem trzech typ贸w miejsc aktywnych, podczas bada艅 bia艂ka zaobserwowano tak偶e kilka 1/2 Streszczenie innych, niespodziewanych zjawisk, kt贸re nie dawa艂y si臋 wyja艣ni膰 w ramach obowi膮zuj膮cego dla enzym贸w paradygmatu, 偶e bia艂ko jest albo aktywne, albo nieaktywne. Spadek aktywno艣ci bia艂ka przechowywanego w -80掳C ma wyra藕nie dwufazowy charakter. Uwolnienie st臋偶enia bia艂ka jako parametru dopasowania modelu wi膮zania do krzywej miareczkowania niezmiennie zwraca艂o niefizycznie niskie warto艣ci. W analizie miareczkowa艅 kalorymetrycznych, gdzie parametrem dopasowania mo偶e by膰 tak偶e st臋偶enie frakcji nieaktywnej bia艂ka, rozumianej jako frakcja bia艂ka niezdolnego wi膮za膰 ligand, uwolnienie tego parametru, jako parametru dopasowania zawsze zwraca艂o warto艣膰 st臋偶enia bia艂ka wi膮偶膮cego wy偶sz膮 od st臋偶enia bia艂ka aktywnego katalitycznie, wyznaczonego na podstawie pomiar贸w aktywno艣ci specyficznej wobec substratu naturalnego. Wszystkie te spostrze偶enia sugerowa艂y konieczno艣膰 zbadania procesu dezaktywacji bia艂ka. Badania takie przeprowadzono i wykryto przynajmniej dwa stany przej艣ciowe, pomi臋dzy bia艂kiem w pe艂ni aktywnym i ca艂kowicie nieaktywnym, niezdolnym do prowadzenia katalizy i do wi膮zania ligand贸w. W pierwszym zidentyfikowanym stanie przej艣ciowym, jeden z trzech dimer贸w staje si臋 nieaktywny katalitycznie wobec naturalnych substrat贸w, natomiast nadal jest w stanie wi膮za膰 ligandy i prowadzi膰 fosforoliz臋 substrat贸w imituj膮cych stan uprotonowany. Pozosta艂e dwa dimery s膮 sprawne katalitycznie, ka偶dy z nich zawiera podjednostk臋, kt贸rej miejsce aktywne przybiera konformacj臋 zamkni臋t膮. Ta forma bia艂ka charakteryzuje si臋 obni偶on膮 aktywno艣ci膮 specyficzn膮 w stosunku do enzymu, w kt贸rym w konformacji zamkni臋tej mo偶e by膰 jedno miejsce aktywne w ka偶dym z trzech dimer贸w. W kolejnym stanie przej艣ciowym 偶adne z miejsc aktywnych nie mo偶e przej艣膰 do konformacji zamkni臋tej, zatem bia艂ko nie katalizuje fosforolizy substrat贸w naturalnych, natomiast katalizuje fosforoliz臋 substrat贸w imituj膮cych stan uprotonowany. Zaproponowano model, kt贸ry wyja艣nia wszystkie zaobserwowane fakty. Pokazano, 偶e PNP z E. coli rzeczywi艣cie jest heksamerem, a nie trimerem niezale偶nie funkcjonuj膮cych dimer贸w, poniewa偶 bez "wsparcia" strukturalnego, jakie zapewniaj膮 s膮siednie podjednostki, dimer nie jest w stanie prowadzi膰 efektywnej katalizy.Purine Nucleoside Phosphorylase (PNP) from Escherichia coli catalysis reversible reaction of phosphorolytic cleavage of purine nucleosides into corresponding purine base and pentose-1-phosphate. The protein is a hexamer, and in apo form all subunits are in the same conformation. Literature data available at the beginning of this work suggested, that structurally, the protein is in fact a trimer of dimers, because active site of particular subunit in a dimer is cocreated by two aminoacids from a neighbouring subunit of the dimer. This suggested, that the minimal catalytic unit in the protein is a dimer. Solution studies data seemed to confirm that hypothesis, as the kinetic towards phosphate ion in non-Michaelis, and is described by Hill coefficient equals 0,5, suggesting strong negative cooperation [1]. Several crystallographic structures of apoenzyme and various complexes, available in the literature, did not explain the mechanism of cooperation signal transduction between subunits or active sites, neither what is the role of co-subunits in catalysis. In 2002, the crystallographic structure was obtained, in which a closed conformation of some active sites was observed, one in each dimer, and the remaining subunits maintained the open conformation of the active site, as in the apo form. Based on this structure, a catalysis mechanism describing the sequence of events in the active site was proposed [2], which would consist of closing the active site with the participation of Arg24. In this conformation of the active site Asp204 residue, in cooperation with Arg217, can protonate nucleoside substrate. This changes the distribution of charge on the purine ring, leads to the destabilization and breakage of the glycosidic bond. The aim of the doctoral thesis was to verify the proposed mechanism of catalysis by investigating active site mutants, and to explain the mechanism and significance of cooperation between hexameric protein subunits. First indication, that cooperation does not only takes place within a dimer, but also between them, were phosphate binding curves, for which a two binding sites model was not sufficient to describe the curves properly, and three binding sites model had to be applied. Three binding site model for phosphate binding was confirmed with several experimental methods. The proposed model assumes that binding of first phosphate ion leads to formation of three kinds of binding sites, one in closed conformation and two in open conformation, but characterised with different affinity to ligands. Thus, three binding constant describing binding of nucleoside should not be surprising. Existence of three kinds of binding sites was observed also in crystal structures. Through destabilisation of contacts between dimers in hexameric ecPNP, free dimers were created, but they turned out to be not stable enough to catalyse effectively. Mechanism proposed in 2002 was confirmed by kinetic studies of active site mutants, Asp204Ala, Arg217Ala and Arg24Ala, towards natural and mimicking protonated state substrates. It was shown that conformational change and protonation of nucleoside are necessary steps of catalysis. On the basis of the conducted research, a mechanism of catalysis was proposed, taking into account the cooperation between the protein subunits in the dimer. Under conditions of substrate saturation, open and closed subunits in dimers catalyse in close cooperation. When a phosphorolysis reaction takes place in the closed subunit, the open subunit has just finished it, the products are released and the next pair of substrates are bound, the subunit is closed and the cycle begins again. This mechanism is known in the literature as flip-flop. In addition to the existence of three types of active sites, several other unexpected phenomena were observed during protein studies, that could not be explained within the framework of the paradigm that the enzyme is either active or inactive. The decrease in the activity of protein stored at -80掳C has a clearly twophase nature. The optimisation of the protein concentration as a parameter of the binding model fit to the titration curve repeatedly returned non-physically low values. In the analysis of calorimetric titrations, where fraction of inactive protein, meaning a protein fraction incapable of binding ligand, can be an adjustable parameter, optimisation of this parameter as a fit parameter always returned the binding protein concentration higher than the concentration of catalytically active protein, determined from specific activity measurements vs. natural substrate. All these observations suggested the need to investigate the process of protein deactivation. Such research were performed and at least two intermediate states were detected between the fully active and completely inactive protein, unable to catalyse and to bind ligands. In the first identified transition state, one of the three dimers becomes catalytically inactive towards natural substrates, while it is still able to bind ligands and carry out phosphorolysis of substrates imitating the protonated state. The other two dimers are catalytically functional and each of them contains a subunit whose active site adopts a closed conformation. This form of the protein is characterized by a reduced specific activity compared to an enzyme, in which one closed site can be present in each of three dimers. In the next transitional state, none of the active sites can adopt a closed conformation, therefore, the protein does not catalyse the phosphorolysis of natural substrates, but it still catalyses phosphorolysis of substrates mimicking protonated state. A model that explains all observed facts was proposed. It was shown, that PNP from E. coli is indeed a hexamer, and not a trimer of independently functioning dimers, because without structural "support" provided by neighbouring subunits, the dimer is unable to catalyse effectively

    New Insights into Active Site Conformation Dynamics of E. coli PNP Revealed by Combined HD Exchange Approach and Molecular Dynamics Simulations.

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    The biologically active form of purine nucleoside phosphorylase (PNP) from Escherichia coli (EC 2.4.2.1) is a homohexamer unit, assembled as a trimer of dimers. Upon binding of phosphate, neighboring monomers adopt different active site conformations, described as open and closed. To get insight into the functions of the two distinctive active site conformations, virtually inactive Arg24Ala mutant is complexed with phosphate; all active sites are found to be in the open conformation. To understand how the sites of neighboring monomers communicate with each other, we have combined H/D exchange (H/DX) experiments with molecular dynamics (MD) simulations. Both methods point to the mobility of the enzyme, associated with a few flexible regions situated at the surface and within the dimer interface. Although H/DX provides an average extent of deuterium uptake for all six hexamer active sites, it was able to indicate the dynamic mechanism of cross-talk between monomers, allostery. Using this technique, it was found that phosphate binding to the wild type (WT) causes arrest of the molecular motion in backbone fragments that are flexible in a ligand-free state. This was not the case for the Arg24Ala mutant. Upon nucleoside substrate/inhibitor binding, some release of the phosphate-induced arrest is observed for the WT, whereas the opposite effects occur for the Arg24Ala mutant. MD simulations confirmed that phosphate is bound tightly in the closed active sites of the WT; conversely, in the open conformation of the active site of the WT phosphate is bound loosely moving towards the exit of the active site. In Arg24Ala mutant binary complex Pi is bound loosely, too

    1.45\AA resolution crystal structure of recombinant PNP in complex with a pM multisubstrate analogue inhibitor bearing one feature of the postulated transition state

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    Low molecular mass purine nucleoside phosphorylases (PNPs, E.C. 2.4.2.1) are homotrimeric enzymes that are tightly inhibited by immucillins. Due to the positive charge on the ribose like part (iminoribitol moiety) and protonation of the N7 atom of the purine ring, immucillins are believed to act as transition state analogues. Over a wide range of concentrations, immucillins bind with strong negative cooperativity to PNPs, so that only every third binding site of the enzyme is occupied (third-of-the-sites binding). 9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) shares with immucillins the protonation of the N7, but not the positive charge on the ribose like part of the molecule. We have previously shown that DFPP-DG interacts with PNPs with subnanomolar inhibition constant. Here, we report additional biochemical experiments to demonstrate that the inhibitor can be bound with the same K(d) ( approximately 190pM) to all three substrate binding sites of the trimeric PNP, and a crystal structure of PNP in complex with DFPP-DG at 1.45A resolution, the highest resolution published for PNPs so far. The crystals contain the full PNP homotrimer in the asymmetric unit. DFPP-DG molecules are bound in superimposable manner and with full occupancies to all three PNP subunits. Thus the postulated third-of-the-sites binding of immucillins should be rather attribute to the second feature of the transition state, ribooxocarbenium ion character of the ligand or to the coexistence of both features characteristic for the transition state. The DFPP-DG/PNP complex structure confirms the earlier observations, that the loop from Pro57 to Gly66 covering the phosphate-binding site cannot be stabilized by phosphonate analogues. The loop from Glu250 to Gln266 covering the base-binding site is organized by the interactions of Asn243 with the Hoogsteen edge of the purine base of analogues bearing one feature of the postulated transition state (protonated N7 position)
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