Synthesis, Characterization And Cholinesterase Inhibitory Activity Of Novel Piperidone Grafted Spiro Heterocycles

Five new series of new spiro-oxindole-pyrrolizine and pyrrolidine hybrids were synthesized employing facile one-pot three-component reaction of linear or cyclic α-amino acids, isatin and various derivatives of N-acryloyl-bis arylidene piperidin-4- ones dipolarophiles. These reactions afforded new mono-spiroheterocycles and bis-spiroheterocycles by changing the ratio of the starting materials from 1:1:1 to 1:2:2 of dipolarophiles, α-amino acid and isatin. These spiro-cycloadducts were further elucidated using elemental analysis, IR, 1-D and 2-D NMR spectroscopy techniques as well as X-Ray crystallographic data. The newly synthesized compounds were also evaluated for their activity against Alzheimer’s disease using Ellman’s colorimetric assay. In this assay the cholinesterase inhibitory activity of the aforementioned compounds were screened in vitro against acetylcholinesterase enzyme (AChE) from electric eel and butyrylcholinesterase enzyme (BChE) from equine serum, which have the major roles in the manifestation and progression of Alzheimer’s disease

Conformational Sub-states and Dynamics in Human Ribonuclease Family

The enzymes of the ribonuclease (RNase) family catalyze the hydrolysis of ribonucleic acid (RNA). There is a wide interest in therapeutic interventions of human RNases (hRNases) due to their critical role in host defense, cancer cell proliferation and neurodegenerative diseases. Development of structure-based therapeutics targeting individual members of a closely related enzyme family is difficult. The structural conservation among hRNases cannot explain a million-fold difference in the catalytic efficiency of these enzymes. We hypothesize, this ambiguity in the structure activity relationship of hRNases can be explained by their dynamical behavior. The rate of substrate turnover in RNases correlates strongly with the rates of conformational dynamics. Moreover, catalytic efficiency is also linked to the ability of an enzyme to sample conformational sub-states that promote specific interaction between reactants at various stages of catalysis. Detailed characterization of the structure-function-dynamics relationship in hRNases can help in determining similarities and differences in the motions associated to catalysis thereby enhancing the possible druggability of this enzyme family. This study investigates the role of functionally relevant conformational sub-states and dynamics in each step of the RNase members catalytic cycle; apo, substrate-bound, the chemical step, and product release. Computer simulations and nuclear magnetic resonance (NMR) relaxation dispersion experiments indicated an increased dynamics in distal loop regions of RNases in their apo state. Similar dynamical patterns were observed within the members of an individual sub-family, while dynamics were different across sub-families. Nucleotide binding properties probed using computer simulations indicated diverse binding preferences in hRNases. Additionally, structural and dynamical properties of hRNases varied significantly with subtle change in temperature. Quasi anharmonic analysis revealed sampling of separate conformational sub-states in product release step of RNases. Further, NMR chemical shift titrations along with the chemical shift projection analysis revealed distinct conformational rearrangements in hRNases upon binding of ligands that mimic cleaved products. Steady-state kinetics experiments showed million-fold difference in the catalytic efficiency of hRNases. Finally, hybrid quantum mechanical/molecular mechanics calculations identified conformational sub-states associated with the chemical step in bovine RNase A. Broadly, these results strongly support our hypothesis that dynamics modulates catalytic efficiencies of structurally related enzyme super-families like RNases

A Computational perspective on the concerted cleavage mechanism of the natural targets of HIV-1 protease.

Doctoral Degree. University of KwaZulu-Natal, Durban.One infectious disease that has had both a profound health and cultural impact on the human race in recent decades is the Acquired Immune Deficiency Syndrome (AIDS) caused by the Human Immunodeficiency Virus (HIV). A major breakthrough in the treatment of HIV-1 was the use of drugs inhibiting specific enzymes necessary for the replication of the virus. Among these enzymes is HIV-1 protease (PR), which is an important degrading enzyme necessary for the proteolytic cleavage of the Gag and Gag-Pol polyproteins, required for the development of mature virion proteins. The mechanism of action of the HIV-1 PR on the proteolysis of these polyproteins has been a subject of research over the past three decades. Most investigations on this subject have been dedicated to exploring the reaction mechanism of HIV-1 PR on its targets as a stepwise general acid-base process with little attention on a concerted model. One of the shortcomings of the stepwise reaction pathway is the existence of more than two TS moieties, which have led to varying opinions on the exact rate-determining step of the reaction and the protonation pattern of the catalytic aspartate group at the HIV-1 PR active site. Also, there is no consensus on the actual recognition mechanism of the natural substrates by the HIV-1 PR. By means of concerted transition state (TS) structural models, the recognition mode and the reaction mechanism of HIV-1 PR with its natural targets were investigated in this present study. The investigation was designed to elucidate the cleavage of natural substrates by HIV-1 PR using the concerted TS model through the application of computational methods to unravel the recognition and reaction process, compute activation parameters and elucidate quantum chemical properties of the system. Quantum mechanics (QM) methods including the density functional theory (DFT) models and Hartree-Fock (HF), molecular mechanics (MM) and hybrid QM/MM were employed to provide better insight in this topic. Based on experience with concerted TS modelling, the six-membered ring TS structure was proposed. Using a small model system and QM methods (DFT and HF), the enzymatic mechanism of HIV-1 PR was studied as a general acid-base model having both catalytic aspartate group participating and water molecule attacking the natural substrate synchronously. The natural substrate scissile bond strength was also investigated via changes of electronic effects. The proposed concerted six-membered ring TS mechanism of the natural substrate within the entire enzyme was studied using hybrid QM/MM; “Our own N-layered Integrated molecular Orbital and molecular Mechanics” (ONIOM) method. This investigation led us to a new perspective in which an acyclic concerted pathway provided a better approach to the subject than the proposed six-membered model. The natural substrate recognition pattern was therefore investigated using the concerted acyclic TS modelling to examine if HIV-1 (South Africa subtype C, C-SA and subtype B) PRs recognize their substrates in the same manner using ONIOM approach. A major outcome in the present investigation is the computational modelling of a new, potentially active, substrate-based inhibitor through the six-membered concerted cyclic TS modelling and a small system. By modelling the entire enzyme—substrate system using a hybrid QM/MM (ONIOM) method, three different pathways were obtained. (1) A concerted acyclic TS structure, (2) a concerted six-membered cyclic TS model and (3) another sixmembered ring TS model involving two water molecules. The activation free energies obtained for the first and the last pathways were in agreement with in vitro HIV-1 PR hydrolysis data. The mechanism that provides marginally the lowest activation barrier involves an acyclic TS model with one water molecule at the HIV-1 PR active site. The outcome of the study provides a plausible theoretical benchmark for the concerted enzymatic mechanism of HIV-1 PRs which could be applied to related homodimeric protease and perhaps other enzymatic processes. Applying the one-step concerted acyclic catalytic mechanism for two HIV-1 PR subtypes, the recognition phenomena of both enzyme and substrate were studied. It was observed that the studied HIV-1 PR subtypes (B and C-SA) recognize and cleave at both scissile and non-scissile regions of the natural substrate sequences and maintaining preferential specificity for the scissile bonds with characteristic lower activation free energies. Future studies on the reaction mechanism of HIV-1 PR and natural substrates should involve the application of advanced computational techniques to provide plausible answers to some unresolved perspectives. Theoretical investigations on the enzymatic mechanism of HIV-1 PR— natural substrate in years to come, would likely involve the application of sophisticated computational techniques aimed at exploring more than the energetics of the system. The possibility of integrated computational algorithms which do not involve partitioning/restraining/constraining/cropped model systems of the enzyme—substrate mechanism would likely surface in future to accurately elucidate the HIV-1 PR catalytic process on natural substrates/ligands

プロリンペプチド結合異性化機構の構造生物学的研究

広島大学(Hiroshima University)博士(理学)Doctor of Sciencedoctora

Strukturelle Dynamik von Peptidyl-Carrier-Domänen in nicht-ribosomalen Peptid-Synthetasen

Eine große Zahl natürlicher sekundärer Metabolite sind kleine und strukturell oft sehr verschiedene Polypeptide und Polyketide. Diese bioaktiven Substanzen haben im allgemeinen ein breit aufgestelltes therapeutisches Potential und werden von verschiedenen bakteriellen Stämmen und Pilzen biosynthetisiert. Sie sind sowohl biologisch, als auch therapeutisch wichtig als Cytostatika, Immunsuppressiva und Antibiotika mit einem sehr großen antibakteriellen und antiviralen Potential. Diese oft äußerst komplexen Polypeptide und Polyketide werden von modular aufgebauten Megaenzymen in mehrstufigen Mechanismen synthetisiert. Für die Synthese dieser Peptide sind sehr große Proteincluster verantwortlich, die meistens aus einer begrenzten Anzahl sehr großer, Multidomänen umfassenden, Superenzyme aufgebaut werden. Diese Proteincluster mit einem Molekulargewicht bis in den Bereich von MegaDalton werden als nicht-ribosomale Peptidsynthetasen (NRPS) und Polyketidsynthetasen (PKS) bezeichnet. Die NRPS Systeme zeichnen sich dadurch aus, daß für die biosynthetisierten Polypeptide keine Information in Form von Nukleinsäuren wie DNA oder RNA kodiert (Walsh, C.T., 2004; Sieber & Marahiel, 2005). Für die Synthese der Polypeptide ist eine Aktivierung der einzelnen Bausteine, der Aminosäuren, durch Amino-acyl-adenylierung notwendig. Im Anschluß an die Aktivierung, wird die aktivierte Aminosäure über einen Thioester gebunden weitertransportiert. Die Thioesterbildung erfolgt an Cysteaminthiolgruppen intrinsischer 4’-Phosphopantethein-kofaktoren. Eine Modul einer NRPS stellt eine geschlossene Einheit zum Einbau einer Aminosäure mit einer hohen Spezifität für das Substrat und die biosynthetische Reaktion dar. Diese Module sind aus Domänen aufgebaut, die definierte Funktionen haben und mittels flexibler Linker miteinander verbunden sind. Die Domänen werden nach ihrer Funktion unterschieden. Die Acyl-adenylierung oder Aktivierung eines Substrates, beispielsweise einer Aminosäure, erfolgt durch die A-Domänen. Die Peptidyl- oder Acyltransportfunktion der aktivierten Substrate wird durch Thioester-domänen (T-Domäne), auch PCP (peptidyl carrier domain) genannt, bewältigt. Die Biosynthese der Kopplungsreaktion, beispielsweise die Ausbildung der Peptidbindung in NRPS Systemen, erfolgt an den Kondensations-Domänen (C-Domäne). Für die Substratspezifität eines Synthesemoduls sind die A-Domänen verantwortlich, welche die Aktivierung eines Substrat durch ATP-Hydrolyse ermöglichen. In NRPS Systemen sind auch Zyklisierungsreaktionen, durchgeführt von Cyclase-Domänen (Cy-Domänen), L/D-Epimerase-funktionen (E-Domänen) und N-Methylierungen (M-Domänen) beschrieben. So wird in Tyrocidin A an zwei Positionen spezifisch Phenylalanin in die D-Form epimerisiert und anschließend in der Peptidbiosynthese verwendet. Die Interaktion und Erkennung zwischen den multi-modularen Superenzymen, zum korrekten Aufbau der kompletten Synthetase, wurden in letzter Zeit Kommunikations-Domänen (COM-Domänen) beschrieben. Wie die aufgebaute Synthetase die korrekte Sequenz der biosynthetischen Reaktionsschritte sicherstellt ist nicht bekannt. Die enorme Diversität biosynthetischer Reaktionen in NRPS Systemen und die hohe Substratvielfalt in den verschiedensten Synthetasen unterschiedlicher Stämme eröffnet ein weites Feld für mögliche Neukombinationen von Modulen und Modifikationen von Produkten, um neue bioaktive Polypeptide mit antibiotischen Eigenschaften durch die Gestaltung neuer biosynthetischer Reaktionswege zu erhalten. Die Biosyntheseprodukte der NRPS und PKS Systeme lassen sich Gruppen kategorisieren wie Peptidantibiotika, beispielsweise beta-Lactame und makrozyklischer Polypeptide. Weitere Gruppen sind die makrozyklischen Lactone, beispielsweise Polyene und Makrolide, aromatische Verbindungen, wie Chloramphenicol, und Chinone (Tetracyclin). Die näher diskutierten Beispiele sind die antibakteriellen Polypeptide Surfactin und Tyrocidin A. Surfactin ist ein antibakteriell wirkendes makrozyklisches Lipoheptapeptid, welches von Bacillus subtilis synthetisiert wird und ein enormes antivirales Potential besitzt. Tyrocidin A ist ein antibakteriell wirkendes makrozyklisches Decapeptid und wird von Bacillus brevis und Brevisbacillus parabrevis synthetisiert. Zusätzlich werden viele bakterielle Toxine ebenfalls durch solche Systeme multi-modularer Synthetasen erzeugt. Ein Beispiel ist das Polyketid Vibriobactin, das Toxin des humanpathogenen Bakterium Vibrio cholerae. Ein zunehmendes Problem der wachsenden Weltbevölkerung moderner Gesellschaften und in den Entwicklungsländern ist die wachsende Zahl multiresistenter Bakterienstämme. Die starke Progression in der Entwicklung von Resistenzen gegen Antibiotika ist auch Gegenstand des aktuellen WHO-Reports (2006). Alarmierend ist die beschleunigte Resistenzentwicklung gegen die sogenannten Reserveantibiotika Vancomycin und Ceftazidim. Ein umfangreicheres Verständnis der Interaktion zwischen Domänen in einem Modul und zwischen Modulen eines NRPS Systems ist Grundlage für die Neukombination unterschiedlicher Module zur erfolgreichen Gestaltung neuer Biosynthesen. Da die meisten dieser Biosynthesen oder die Synthese alternativer Substanzen nicht in der Organischen Chemie zu realisieren sind oder die Produkte zu teuer wären, um diese in großen Mengen zu erzeugen, muß das Ziel sein die NRPS und PKS Systeme in ihrem modularen Aufbau und ihre Interaktion zu verstehen, um alternative Antibiotika biosynthetisch herzustellen. Peptidyl Carrier Proteine (PCPs) sind kleine zentrale Transport-Domänen, integriert in den Modulen nicht-ribosomaler Peptidsynthetasen (NRPSs). PCPs tragen kovalent über eine Phosphoesterbindung einen aus dem Protein herausragenden 4’-phosphopantetheinyl (4’-PP) Kofaktor. Der 4’-PP Kofaktor ist an der Seitenkette eines hochkonservierten Serins gebunden, welche ein zentraler Bestandteil der Phosphopantethein-Erkennungs-Sequenz ist. Die Erkennungssequenz ist homolog in vielen Proteinen mit ähnlicher Funktion, inklusive Acyl Carrier Proteinen (ACPs) der Fettsäuresynthetasen (FAS) und der Polyketidsynthetasen (PKS). Die Thiolgruppe des 4’-PP Kofaktors dient zum aktiven Transport der Substrate und der Intermediate der NRPS Systeme. Die generelle Organisation und die Kontrolle der exakt aufeinander folgenden Reaktionsschritte in der Peptidsynthetase, ist die entscheidende Frage für die Funktion des Proteinclusters (assembly line mechanism). In Modulen der NRPS Systeme folgen die PCP-Domänen C-terminal auf die Adenylierungsdomänen (A-Domäne). Die Aufgabe der A-Domänen ist die Selektion and die Aktivierung einer spezifischen Aminosäure für die „assembly line“. Die eigentliche Bildung der Peptidbindung erfolgt an der Kondensations-Domäne (C-Domäne). Der Transfer der Peptidintermediate und der aktivierten Aminosäuren zwischen A-Domänen und C-Domänen ist Aufgabe der PCPs. Um diese Funktion erfüllen zu können, ist eine große Bewegung in PCPs, bzw. des 4’-PP Kofaktors notwendig, welche als „swinging arm model“ (Weber et al., 2001) beschrieben wurde. Die PCPs koordinieren damit die Peptidbiosynthese während sie mit diversen Domänen der Synthetasen spezifisch wechselwirken müssen. Die molekularen Mechanismen des Transportes wurden bisher allerdings nicht untersucht. Eine Dynamik der Transport-Domänen wurde bereits postuliert (Kim & Prestegard, 1989; Andrec et al., 1995), konnte bisher aber nicht gezeigt werden (Weber et al., 2001). Interessanterweise zeigt sowohl apo-PCP (ohne den kovalent gebundenen 4’-PP Kofaktor) also auch holo-PCP langsamen chemischen Austausch, der als jeweils zwei stabile Konformationen beschrieben werden konnte. Diese jeweils zwei stabilen Zustände, welche sich im Austausch befinden, wurden als A und A*, für apo-PCP, und entsprechend H und H* für holo-PCP bezeichnet. Während der A- und der H-Zustand sich sowohl voneinander als auch von den entsprechenden A* und H*-Zuständen unterscheiden und spezifisch für die apo- und die holo-Form von PCP sind, ist die kalkulierte Struktur vom A*-Zustand größten Teils identisch mit der des H*-Zustandes. Die erhaltenen NMR-Strukturen des A-Zustandes, des H-Zustandes und des gemeinsamen A/H-Zustandes beschreiben in ihrer Gesamtheit ein neues Modell für ein allosterie-kontrolliertes System dualer konformationeller Zwei-Zustands-Dynamik. Zu dem beobachteten konformationellen Austausch der PCP-Domäne, konnte die Bewegung des 4’-PP Kofaktors koordiniert werden. Die Bewegung des 4’-PP Kofaktors in Verbindung mit dem konformationellen Austausch der PCP-Domäne charakterisiert die Interaktion mit katalytischen Domänen eines NRPS Moduls. Des weiteren konnte mit Hilfe des Modells die Wechselwirkung mit externen Interaktionspartnern, wie der Thioesterase II und der 4’-PP Transferase, untersucht werden. Die externe Thioesterase II der Surfactin-Synthetase (SrfTEII) von Bacillus subtilis ist ein separat expremiertes 28 KDa Protein. Sie gehört zur Familie der alpha/beta-Hydrolasen und ist verantwortlich für die Regenerierung falsch beladener 4’- PP Kofaktoren der Peptidyl Carrier Domänen. Die SrfTEII wurde mittels Lösungs-NMR untersucht, die Resonanzen wurden zugeordnet, erste strukturelle Modelle konnte berechnet werden und das Interaktionsverhalten mit verschiedenen modifizierten Kofaktoren und PCPs wurde analysiert. Die Spezifität der Substraterkennung durch die SrfTEII kann beschrieben werden. Interessanterweise zeigt auch die SrfTEII Doppelpeaks für einzelne Aminosäuren, diese können als Indikator für eine spezifische Substraterkennung durch das Enzym verwendet werden und helfen den funktionellen Unterschied zwischen der SrfTEI-Domäne und SrfTEII zu verstehen

The mechanistic modelling of HIV-1 protease and its natural substrates: a theoretical perspective.

Doctoral Degree. University of KwaZulu-Natal, Durban.An epidemic that has had profound impact on humanity both culturally and health-wise in recent decades is the Acquired immunodeficiency syndrome (AIDS) triggered by the Human immunodeficiency virus (HIV). The developments of drugs, impeding specific enzymes essential for the replication of the HIV-1 virus, has been a breakthrough in the treatment of the virus. These enzymes include the HIV-1 protease (PR), which is a significant degrading enzyme necessary for the proteolytic cleavage of the Gag and Gag-Pol polyproteins, needed for the maturation of viral protein. The catalytic mechanism of the HIV-1 PR of these polyproteins is a major subject of investigation over the past decades. Most research on this topic explores the HIV-1 PR mechanism of action on its target as a stepwise general acid-base mechanism with little or no attention to the concerted process. Among the limitations of the stepwise reaction model is the presence of more than two transition state (TS) steps and this led to different views on the precise rate-determining step of the reaction as well as the protonation state of the catalytic aspartate in the active site of the HIV-1 PR. Likewise, consensus on the exact recognition mechanism of the natural substrates by HIV-1 PR is not forthcoming. The present study investigates the recognition approach and mechanism of reaction of the HIV-1 PR with its natural substrate by a means of computational models. It is intended to explain the cleavage mechanism of the reaction as a concerted process through the application of in-silico techniques. This is achieved by computing the activation energies and elucidating the quantum chemical properties of the enzyme-substrate system. An improved understanding of the mechanism will assist in the design of new HIV-1 PR inhibitors. The molecular dynamics (MD) technique with hybrid quantum mechanics and molecular mechanics (QM/MM) method that includes the density functional theory (DFT) and Amber model were utilized to investigate the concerted hydrolysis process. Based on previous studies in our group involving concerted TS modeling, a six-membered ring TS pathway was first considered. This was achieved by employing a small model system and QM methods (Hartree-Fock and DFT) for the enzymatic mechanism of the HIV-1 PR. A general-acid base (GA/GB) model where both catalytic aspartate (Asp) groups are involved in the mechanism, and the water molecule at the active site attacks the natural substrate synchronously, was utilized. A new perspective arose from the study where an acyclic concerted computational model offered activation energies closer to experiment observations than the six-membered ring model. Hence, the proposed concerted acyclic mechanism of the HIV-1 natural substrate within the entire protease was investigated using both multi-layered QM/MM “Our own N-layered Integrated molecular Orbital and molecular Mechanics” (ONIOM) theory and QM/MM MD umbrella sampling method. A comprehensive review about experimental and theoretical results for the interactions between HIV PR and its natural substrates was presented. An important output in the present study is that the acyclic TS model barrier with one water molecule at the HIV-1 PR active site (DFT study), provides marginally, the most accurate activation energies. Similarly, the computational model demonstrated that optimum recognition specificity of the enzyme depends on structural details of the substrates as well as the number of amino acids in the substrate sequence (minimum P5-P5ʹ required). By modelling the entire enzyme—substrate system using a hybrid ONIOM QM/MM method, it was observed that although both subtype B and C-SA HIV-1 PR recognize and cleave at the scissile and non-scissile regions of the natural substrate sequence, the scissile region has a lower activation free energy. In all cases we found activation free energies that are in good agreement with experimental results. Also, the free energy profiles obtained from the umbrella sampling model were in absolute agreement with experimental in vitro HIV-1 PR hydrolysis data. The outcome of this investigations offers a plausible theoretical yardstick for the concerted enzymatic mechanism of the HIV-1 PRs that is pragmatic to related aspartate proteases and possibly other enzymatic processes. Future studies on the reaction mechanism of HIV-1 PR and its natural substrate should encompass the use of advanced theoretical techniques aimed at exploring more than the energetics of the system. The prospect of integrated computational algorithms that does not involve cropped/partitioning/constraining or restraining model systems of the enzyme—substrate mechanism to accurately elucidate the HIV-1 PR catalytic process on natural substrates/inhibitors will be undertaken in our group. Computational investigations on the enzymatic mechanism of the HIV-1 PR—natural substrate involves fine-tuning the scissile amide bond strength through steric and electronic factors. This may lead to the development of potential substrate-based inhibitors with better potency and reduced toxicity. ISIQEPHU Ubhubhane olube nomthelela omkhulu ebuntwini bobabili ngokwemvelo nangokuqonda kwezempilo emashumini eminyaka amuva nje yi-Acquired immunodeficiency syndrome (AIDS) okubangelwa yi-Human immunodeficiency virus (HIV). Ukuthuthuka kwezidakamizwa, okufaka amandla ama-enzyme athile abalulekile ekuphindaphindweni kwegciwane le-HIV-1, kube yimpumelelo ekwelashweni kwaleli gciwane. La ma-enzyme afaka i-HIV-1 proteinase (PR), okuyi-enzyme ebalulekile eyonakalisayo edingekayo ekuhlanzeni kwe-protein ye-Gag ne-GagPol, edingeka ekuvuthweni kweprotheni yegciwane. Indlela ebusayo ye-HIV-1 PR yalezi zipolyprotein iyinto enkulu ephenywayo emashumini eminyaka edlule. Ucwaningo oluningi ngalesi sihloko luhlola indlela esebenza ngayo ye-HIV-1 PR kulokho okukuhlosile njengenyathelo elisisekelo le-acid-base elisebenzayo ngaphandle kokunaka noma lengenayo inqubo ehlanganisiwe. Phakathi kokukhawulelwa kwemodeli yokusabela esezingeni eliphansi kukhona ubukhona bezinyathelo ezingaphezu kwezimbili zokuguqula isimo (TS) futhi lokhu kuholele ekubukweni okuhlukile esilinganisweni esinqunyiwe sokulinganisa sokuphendula kanye nesimo sokuhlasela sethonya elishukumisayo kulowo osebenzayo indawo ye-HIV-1 PR. Ngokunjalo, ukuvumelana mayelana nendlela ngqo yokuqashelwa kwezakhi zemvelo nge-HIV-1 PR akusondeli. Ucwaningo lwamanje luphenya indlela yokuqashelwa kanye nendlela yokusabela kwe-HIV-1 PR ngesakhiwo sayo esingokwemvelo ngezindlela zamamodeli wokuncintisana. Kuhloswe ukuchaza indlela ye-cleavage yokusabela njengenqubo ekhonjiwe ngokusebenzisa amasu we-in-silico. Lokhu kutholakala ngokuhlanganisa amandla we-activation amandla kanye nokucacisa izakhiwo zamakhemikhali we-quantum wohlelo lwangaphansi lwe-enzyme. Ukuqonda okungcono kwendlela ezokusiza ekwakhiweni kwama-inhibitors amasha we-HIV-1 PR. Indlela esetshenziswayo yama-molecule (i-MD) ene-hybrid quantum mechanics kanye nemolecule mechanics (QM / MM) efaka inqubo yokusizakala yokusebenza kwe-density theory (DFT) kanye ne-Amber model ukuphenya inqubo ekhonjiwe ye-hydrolysis. Ngokusekelwe kwizifundo zangaphambili eqenjini lethu ezibandakanya ukumodelwa kwe-TS ekhonjiwe, indlela eyindilinga eyisithupha yomgwaqo eyi-TS yaqala ukubhekwa. Lokhu kutholwe ngokusebenzisa uhlelo olusha lwemodeli nezindlela ze-QM (Hartree-Fock ne-DFT) ngomshini we-enzymatic we- HIV-1 PR. Imodeli ejwayelekile ye-acid-(GA / GB) lapho amaqembu womabili we-catalytic aspartate (Asp) abandakanyeka khona emshinini, futhi i-molecule lamanzi esakhiweni esisebenzayo lihlasela i-substrate yemvelo ngokuvumelanisa, lalisetshenziswa. Kuqhamuke umbono omusha ocwaningweni lapho imodeli ye-acyclic ekhonjiwe yokuhlinzekwa kwamandla inika amandla okusebenzisa eduze nokuhlolwa okubonwayo kunasekuqaleni kwendandatho eyindandatho eyisithupha. Ngakho-ke, indlela ehlongozwayo ekhonjwe ngendlela ekhanyayo yeHIV-1 substrate yemvelo kuyo yonke iprotease iphenyisisiwe kusetshenziswa ama-QM / MM amaningi ahlukaniswe ngama-Mechanics”(ONIOM) kanye ne-QM / MM MD isampula isambulela indlela. Ukubuyekezwa okuphelele mayelana nemiphumela yokulinga kanye nemibhalo theory yokuxhumana phakathi kwe-HIV PR nezakhi zayo zemvelo kwalethwa. Umphumela obalulekile ocwaningweni lwamanje ukuthi isithintelo se-acyclic TS imodeli nge-mocule eyodwa yamanzi kwisiza esisebenzayo se-HIV-1 PR (i-DFT), sinikela ngamandla, amandla anembe kakhulu okusebenza. Ngokufanayo, imodeli yokuhlanganisa ibonise ukuthi ukuqashelwa okuphelele kweenzyme kuncike kwimininingwane yokwakheka kwama-substrates kanye nenani lama-amino acid ngokulandelana kwe-substrate (ubuncane be-P5-P5'). Ngokumodela yonke i-enzyme — uhlelo olusebenzisa uhlelo lwe-hybrid ONIOM QM / MM, kwaqapheleka ukuthi yize zombili izifunda ezingaphansi kwe-B ne-C-SA ye-HIV-1 PR zibona futhi zinamathele ezindaweni ezibucayi nezingasontekile zendlela yokulandelana engokwemvelo. isifunda esinomswakama sinamandla aphansi we-activation mahhala. Kuzo zonke izimo sithole amandla we-activation mahhala avumelane kahle nemiphumela yokuhlolwa. Futhi, amaphrofayili wamandla wamahhala atholakala kusampuli yesampuli ye-umbrella ayesesivumelwaneni ngokuphelele nedatha yokuhlolwa kwe-vitro HIV-1 PR hydrolysis. Umphumela walolu phenyo uhlinzeka ngokungenaphutha kwethiyori eyingqophamlando ye-enzymatic mechanism ye-HIV-1 PRs edlulele kumaphrotheni ahlobene ne-aspartate kanye nezinye izinqubo ze-enzymatic. Izifundo zesikhathi esizayo mayelana nendlela yokusebenza kwe-HIV-1 PR kanye nengxenye yayo yemvelo kufanele ifake phakathi ukusetshenziswa kwamasu athuthukile we-theorytical okuhloswe ngawo ukuthola ngaphezu komfutho we-system. Ithemba lama-algorithms ahlanganisiwe wokubandakanya okungabandakanyanga okuhlanganisiwe / ukwahlukanisa / ukuphoqelela noma ukuvimba izindlela eziyimodeli ze-enzyme-inqubo engaphansi yokwengeza ukucacisa ngokunembile inqubo yokulwa ne-HIV-1 PR kuzakhi zangaphansi zemvelo / ezinqandweni kuzokwenziwa eqenjini lethu. Uphenyo lwe-computational mayelana ne-enzymatic mechanism ye-HIV-1 PR-substrate yemvelo ifaka phakathi ukulungisa kahle amandla e-bond ayisihlanganisi nge-steric ne-elekthronikhi. Lokhu kungaholela ekwakhiweni kwama-inhibitors angaphansi komhlaba angaphansi nge-potency engcono nokunciphisa ubuthi

Kinetic landscape of a peptide-bond-forming prolyl oligopeptidase

We thank Dr. Rafael Guimaraes da Silva for helpful discussions on enzyme kinetics. We also thank Professor David Lilley, Dr. Alasdair Freeman and Dr. Anne-Cecile Declais at the University of Dundee for training and usage of their QFM-4000 quenched-flow apparatus.Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of this enzyme family is not available. Macrocyclase enzymes are currently exploited to produce cyclic peptides with potential therapeutic applications. Cyclic peptides are promising drug-like molecules due to their stability and conformational rigidity. Here we describe an in-depth kinetic characterization of a prolyl oligopeptidase acting as a macrocyclase enzyme. By combining steady-state and pre-steady-state kinetics, we put forward a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where cyclic peptide departs first followed by the peptide tail. Dissociation of the peptide tail is slow and significantly contributes to the turnover rate. Furthermore, trapping of the enzyme by the peptide tail becomes significant beyond initial-rate conditions. The presence of a burst of product formation and a large viscosity effect further support the rate-limiting nature of a physical step occurring after macrocyclization. This is the first detailed description of the kinetic sequence of a macrocyclase enzyme from this class. GmPOPB is amongst the fastest macrocyclases described to date, and this work is a necessary step towards designing broad specificity efficient macrocyclases.Publisher PDFPeer reviewe

42nd Rocky Mountain Conference on Analytical Chemistry

Abstracts from the 42nd annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-sponsored by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Broomfield, Colorado, July 30 - August 3, 2000