Klonierung, heterologe Expression, Kristallisation und Struktur der Perakin-Reduktase aus Rauvolfia serpentina

Die Perakin-Reduktase (PR) ist ein hochspezifisches Enzym aus dem Alkaloidstoffwechsel in Rauvolfia serpentina, dessen enzymatischer und molekularer Reaktionsmechanismus noch immer unbekannt ist. Um die dreidimensionale Struktur der PR aufzuklären, wurde in der vorliegenden Arbeit das für die PR codierende Gen erstmals identifiziert, exprimiert und das Genprodukt zur Kristallisation gebracht. Die PR ist ein 337 Aminosäure langes monomeres Protein mit einem Molekulargewicht von 37,2 kDa. Die Reinigung erfolgte über Ni2+-NTA-Affinitätschromatographie und lieferte 10 mg homogenes Protein pro Liter Bakterienkultur. Nach Expression in E. coli wurde im Enzym-Assay die NADPH2-abhängige Reduktion von Perakin zu Raucaffrinolin bestätigt und das Endprodukt massenspektrometrisch identifiziert. Durch Sequenzalignments mit anderen Proteinen wurde geschlossen, dass die PR zu der Superfamilie der Aldo/Keto-Reduktasen (AKR) gehört. Nach heterologer Expression in E. coli konnte die homogene, über reduktive Methylierung modifizierte PR mit Hilfe der Methode der Dampfdiffusion im hängenden Tropfen kristallisiert werden. In Gegenwart von 27% PEG 4000 und 100 mM Natriumcitrat (pH 5,6) bildeten sich nach 4 Tagen bei 20°C die ersten Kristalle. Die Struktur der PR konnte mit einer Auflösung von 2,0 Å durch molekularen Ersatz vollständig gelöst werden. Das Strukturmodell besitzt eine für AKRs charakteristische (α/β)8 TIM-barrel Faltung, konservierte Aminosäuren, die an der Bindung von NADPH2 beteiligt sind sowie eine katalytische Tetrade, die den Wasserstofftransfer von NADPH2 zum Kohlenstoff des Substrates vermittelt.

Biochemical analysis of the phosphatase domain of the human soluble epoxide hydrolase (sEH)

Die lösliche Epoxidhydrolase (sEH) gehört zur Familie der Epoxidhydrolase-Enzyme. Die Rolle der sEH besteht klassischerweise in der Detoxifikation, durch Umwandlung potenziell schädlicher Epoxide in deren unschädliche Diol-Form. Hauptsächlich setzt die sEH endogene, der Arachidonsäure verwandte Signalmoleküle, wie beispielsweise die Epoxyeicosatrienoic acid, zu den entsprechenden Diolen um. Daher könnte die sEH als ein Zielenzym in der Therapie von Bluthochdruck und Entzündungen sowie diverser anderer Erkrankungen eingesetzt werden. rnDie sEH ist ein Homodimer, in dem jede Untereinheit aus zwei Domänen aufgebaut ist. Das katalytische Zentrum der Epoxidhydrolaseaktivität befindet sich in der 35 kD großen C-terminalen Domäne. Dieser Bereich der sEH s wurde bereits im Detail untersucht und nahezu alle katalytischen Eigenschaften des Enzyms sowie deren dazugehörige Funktionen sind in Zusammenhang mit dieser Domäne bekannt. Im Gegensatz dazu ist über die 25 kD große N-terminale Domäne wenig bekannt. Die N-terminale Domäne der sEH wird zur Haloacid Dehalogenase (HAD) Superfamilie von Hydrolasen gezählt, jedoch war die Funktion dieses N-terminal Domäne lange ungeklärt. Wir haben in unserer Arbeitsgruppe zum ersten Mal zeigen können, dass die sEH in Säugern ein bifunktionelles Enzym ist, welches zusätzlich zur allgemein bekannten Enzymaktivität im C-terminalen Bereich eine weitere enzymatische Funktion mit Mg2+-abhängiger Phosphataseaktivität in der N-terminalen Domäne aufweist. Aufgrund der Homologie der N-terminalen Domäne mit anderen Enzymen der HAD Familie wird für die Ausübung der Phosphatasefunktion (Dephosphorylierung) eine Reaktion in zwei Schritten angenommen.rnUm den katalytischen Mechanismus der Dephosphorylierung weiter aufzuklären, wurden biochemische Analysen der humanen sEH Phosphatase durch Generierung von Mutationen im aktiven Zentrum mittels ortsspezifischer Mutagenese durchgeführt. Hiermit sollten die an der katalytischen Aktivität beteiligten Aminosäurereste im aktiven Zentrum identifiziert und deren Rolle bei der Dephosphorylierung spezifiziert werden. rnrnAuf Basis der strukturellen und möglichen funktionellen Ähnlichkeiten der sEH und anderen Mitgliedern der HAD Superfamilie wurden Aminosäuren (konservierte und teilweise konservierte Aminosäuren) im aktiven Zentrum der sEH Phosphatase-Domäne als Kandidaten ausgewählt.rnVon den Phosphatase-Domäne bildenden Aminosäuren wurden acht ausgewählt (Asp9 (D9), Asp11 (D11), Thr123 (T123), Asn124 (N124), Lys160 (K160), Asp184 (D184), Asp185 (D185), Asn189 (N189)), die mittels ortsspezifischer Mutagenese durch nicht funktionelle Aminosäuren ausgetauscht werden sollten. Dazu wurde jede der ausgewählten Aminosäuren durch mindestens zwei alternative Aminosäuren ersetzt: entweder durch Alanin oder durch eine Aminosäure ähnlich der im Wildtyp-Enzym. Insgesamt wurden 18 verschiedene rekombinante Klone generiert, die für eine mutante sEH Phosphatase Domäne kodieren, in dem lediglich eine Aminosäure gegenüber dem Wildtyp-Enzym ersetzt wurde. Die 18 Mutanten sowie das Wildtyp (Sequenz der N-terminalen Domäne ohne Mutation) wurden in einem Expressionsvektor in E.coli kloniert und die Nukleotidsequenz durch Restriktionsverdau sowie Sequenzierung bestätigt. Die so generierte N-terminale Domäne der sEH (25kD Untereinheit) wurde dann mittels Metallaffinitätschromatographie erfolgreich aufgereinigt und auf Phosphataseaktivität gegenüber des allgemeinen Substrats 4-Nitophenylphosphat getestet. Diejenigen Mutanten, die Phosphataseaktivität zeigten, wurden anschließend kinetischen Tests unterzogen. Basiered auf den Ergebnissen dieser Untersuchungen wurden kinetische Parameter mittels vier gut etablierter Methoden berechnet und die Ergebnisse mit der „direct linear blot“ Methode interpretiert. rnDie Ergebnisse zeigten, dass die meisten der 18 generierten Mutanten inaktiv waren oder einen Großteil der Enzymaktivität (Vmax) gegenüber dem Wildtyp verloren (WT: Vmax=77.34 nmol-1 mg-1 min). Dieser Verlust an Enzymaktivität ließ sich nicht durch einen Verlust an struktureller Integrität erklären, da der Wildtyp und die mutanten Proteine in der Chromatographie das gleiche Verhalten zeigten. Alle Aminosäureaustausche Asp9 (D9), Lys160 (K160), Asp184 (D184) und Asn189 (N189) führten zum kompletten Verlust der Phosphataseaktivität, was auf deren katalytische Funktion im N-terminalen Bereich der sEH hindeutet. Bei einem Teil der Aminosäureaustausche die für Asp11 (D11), Thr123 (T123), Asn124 (N124) und Asn185 (D185) durchgeführt wurden, kam es, verglichen mit dem Wildtyp, zu einer starken Reduktion der Phosphataseaktivität, die aber dennoch für die einzelnen Proteinmutanten in unterschiedlichem Ausmaß zu messen war (2 -10% and 40% of the WT enzyme activity). Zudem zeigten die Mutanten dieser Gruppe veränderte kinetische Eigenschaften (Vmax allein oder Vmax und Km). Dabei war die kinetische Analyse des Mutanten Asp11  Asn aufgrund der nur bei dieser Mutanten detektierbaren starken Vmax Reduktion (8.1 nmol-1 mg-1 min) und einer signifikanten Reduktion der Km (Asp11: Km=0.54 mM, WT: Km=1.3 mM), von besonderem Interesse und impliziert eine Rolle von Asp11 (D11) im zweiten Schritt der Hydrolyse des katalytischen Zyklus.rnZusammenfassend zeigen die Ergebnisse, dass alle in dieser Arbeit untersuchten Aminosäuren für die Phosphataseaktivität der sEH nötig sind und das aktive Zentrum der sEH Phosphatase im N-terminalen Bereich des Enzyms bilden. Weiterhin tragen diese Ergebnisse zur Aufklärung der potenziellen Rolle der untersuchten Aminosäuren bei und unterstützen die Hypothese, dass die Dephosphorylierungsreaktion in zwei Schritten abläuft. Somit ist ein kombinierter Reaktionsmechanismus, ähnlich denen anderer Enzyme der HAD Familie, für die Ausübung der Dephosphorylierungsfunktion denkbar. Diese Annahme wird gestützt durch die 3D-Struktur der N-terminalen Domäne, den Ergebnissen dieser Arbeit sowie Resultaten weiterer biochemischer Analysen. Der zweistufige Mechanismus der Dephosphorylierung beinhaltet einen nukleophilen Angriff des Substratphosphors durch das Nukleophil Asp9 (D9) des aktiven Zentrums unter Bildung eines Acylphosphat-Enzym-Zwischenprodukts, gefolgt von der anschließenden Freisetzung des dephosphorylierten Substrats. Im zweiten Schritt erfolgt die Hydrolyse des Enzym-Phosphat-Zwischenprodukts unterstützt durch Asp11 (D11), und die Freisetzung der Phosphatgruppe findet statt. Die anderen untersuchten Aminosäuren sind an der Bindung von Mg 2+ und/oder Substrat beteiligt. rnMit Hilfe dieser Arbeit konnte der katalytischen Mechanismus der sEH Phosphatase weiter aufgeklärt werden und wichtige noch zu untersuchende Fragestellungen, wie die physiologische Rolle der sEH Phosphatase, deren endogene physiologische Substrate und der genaue Funktionsmechanismus als bifunktionelles Enzym (die Kommunikation der zwei katalytischen Einheiten des Enzyms) wurden aufgezeigt und diskutiert.rn

The synthesis of maleic anhydride: study of a new process and improvement of the industrial catalyst

Maleic anhydride is an important chemical intermediate mainly produced by the selective oxidation of n-butane, an industrial process catalyzed by vanadyl pyrophosphate-based materials, (VO)2P2O7. The first topic was investigated in collaboration with a company specialized in the production of organic anhydrides (Polynt SpA), with the aim of improving the performance of the process for the selective oxidation of n-butane to maleic anhydride, comparing the behavior of an industrial vanadyl pyrophosphate catalysts when utilized either in the industrial plant or in lab-scale reactor. The study was focused on how the catalyst characteristics and reactivity are affected by the reaction conditions and how the addition of a dopant can enhance the catalytic performance. Moreover, the ageing of the catalyst was studied, in order to correlate the deactivation process with the modifications occurring in the catalyst. The second topic was produced within the Seventh Framework (FP7) European Project “EuroBioRef”. The study was focused on a new route for the synthesis of maleic anhydride starting from an alternative reactant produced by fermentation of biomass:“bio-1-butanol”. In this field, the different possible catalytic configurations were investigated: the process was divided into two main reactions, the dehydration of 1-butanol to butenes and the selective oxidation of butenes to maleic anhydride. The features needed to catalyze the two steps were analyzed and different materials were proposed as catalysts, namely Keggin-type polyoxometalates, VOPO4∙2H2O and (VO)2P2O7. The reactivity of 1-butanol was tested under different conditions, in order to optimize the performance and understand the nature of the interaction between the alcohol and the catalyst surface. Then, the key intermediates in the mechanism of 1-butanol oxidehydration to MA were studied, with the aim of understanding the possible reaction mechanism. Lastly, the reactivity of the chemically sourced 1-butanol was compared with that one of different types of bio-butanols produced by biomass fermentation.

Selective oxidation of 5-hydroxymethylfurfural using monometallic and bimetallic supported nanoparticles

This work deals with the oxidation of 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA) using metal supported catalysts. Catalysts were prepared from the immobilisation of preformed monometallic (Au, Pd) and bimetallic (AuCu, AuPd) nanoparticles on commercial oxides (TiO2, CeO2). Au-TiO2 catalyst was found to be very active for HMF oxidation; however, this system deactivated very fast. For this reason, we prepared bimetallic gold-copper nanoparticles and an increase in the catalytic activity was observed together with an increase in catalyst stability. In order to optimise the interaction of the metal active phase with the support, Au and AuCu nanoparticles were supported onto CeO2. Au-CeO2 catalyst was found to be more active than the bimetallic one, leading to the conclusion that in this case the most important feature is the interaction between gold and the support. Catalyst pre-treatments (calcination and washing) were carried out to maximise the contact between the metal and the oxide and an increase in the FDCA production could be observed. The presence of ceria defective sites was crucial for FDCA formation. Mesoporous cerium oxide was synthesised with the hard template method and was used as support for Au nanoparticles to promote the catalytic activity. In order to study the role of active phase in HMF oxidation, PdAu nanoparticles were supported onto TiO2. Au and Pd monometallic catalysts were very active in the formation of HMFCA (5-hydroxymethyl-2-furan carboxylic acid), but Pd was not able to convert it, leading to a low FDCA yield. The calcination of PdAu catalysts led to Pd segregation on the particles surface, which changed the reaction pathway and included an important contribution of the Cannizzaro reaction. PVP protected PdAu nanoparticles, synthesised with different morphologies (core-shell and alloyed structure), confirmed the presence of a different reaction mechanism when the metal surface composition changes.

Formation and surface exchange of nitrous acid

Die salpetrige Säure (HONO) ist eine der reaktiven Stickstoffkomponenten der Atmosphäre und Pedosphäre. Die genauen Bildungswege von HONO, sowie der gegenseitige Austausch von HONO zwischen Atmosphäre und Pedosphäre sind noch nicht vollständig aufgedeckt. Bei der HONO-Photolyse entsteht das Hydroxylradikal (OH) und Stickstoffmonooxid (NO), was die Bedeutsamkeit von HONO für die atmosphärische Photochemie widerspiegelt.rnUm die genannte Bildung von HONO im Boden und dessen anschließenden Austausch mit der Atmosphäre zu untersuchen, wurden Messungen von Bodenproben mit dynamischen Kammern durchgeführt. Im Labor gemessene Emissionsflüsse von Wasser, NO und HONO zeigen, dass die Emission von HONO in vergleichbarem Umfang und im gleichen Bodenfeuchtebereich wie die für NO (von 6.5 bis 56.0 % WHC) stattfindet. Die Höhe der HONO-Emissionsflüsse bei neutralen bis basischen pH-Werten und die Aktivierungsenergie der HONO-Emissionsflüsse führen zu der Annahme, dass die mikrobielle Nitrifikation die Hauptquelle für die HONO-Emission darstellt. Inhibierungsexperimente mit einer Bodenprobe und die Messung einer Reinkultur von Nitrosomonas europaea bestärkten diese Theorie. Als Schlussfolgerung wurde das konzeptionelle Model der Bodenemission verschiedener Stickstoffkomponenten in Abhängigkeit von dem Wasserhaushalt des Bodens für HONO erweitert.rnIn einem weiteren Versuch wurde zum Spülen der dynamischen Kammer Luft mit erhöhtem Mischungsverhältnis von HONO verwendet. Die Messung einer hervorragend charakterisierten Bodenprobe zeigte bidirektionale Flüsse von HONO. Somit können Böden nicht nur als HONO-Quelle, sondern auch je nach Bedingungen als effektive Senke dienen. rnAußerdem konnte gezeigt werden, dass das Verhältnis von HONO- zu NO-Emissionen mit dem pH-Wert des Bodens korreliert. Grund könnte die erhöhte Reaktivität von HONO bei niedrigem pH-Wert und die längere Aufenthaltsdauer von HONO verursacht durch reduzierte Gasdiffusion im Bodenporenraum sein, da ein niedriger pH-Wert mit erhöhter Bodenfeuchte am Maximum der Emission einhergeht. Es konnte gezeigt werden, dass die effektive Diffusion von Gasen im Bodenporenraum und die effektive Diffusion von Ionen in der Bodenlösung die HONO-Produktion und den Austausch von HONO mit der Atmosphäre begrenzen. rnErgänzend zu den Messungen im Labor wurde HONO während der Messkampagne HUMPPA-COPEC 2010 im borealen Nadelwald simultan in der Höhe von 1 m über dem Boden und 2 bis 3 m über dem Blätterdach gemessen. Die Budgetberechnungen für HONO zeigen, dass für HONO sämtliche bekannte Quellen und Senken in Bezug auf die übermächtige HONO-Photolyserate tagsüber vernachlässigbar sind (< 20%). Weder Bodenemissionen von HONO, noch die Photolyse von an Oberflächen adsorbierter Salpetersäure können die fehlende Quelle erklären. Die lichtinduzierte Reduktion von Stickstoffdioxid (NO2) an Oberflächen konnte nicht ausgeschlossen werden. Es zeigte sich jedoch, dass die fehlende Quelle stärker mit der HONO-Photolyserate korreliert als mit der entsprechenden Photolysefrequenz, die proportional zur Photolysefrequenz von NO2 ist. Somit lässt sich schlussfolgern, dass entweder die Photolyserate von HONO überschätzt wird oder dass immer noch eine unbekannte, HONO-Quelle existiert, die mit der Photolyserate sehr stark korreliert. rn rn

Complessi carbenici N-eterociclici di rutenio: catalizzatori omogenei in reazioni di idrogenazione e deidrogenazione

Il presente lavoro di tesi si inserisce in un progetto di ricerca volto alla sintesi di nuovi complessi di metalli di transizione per lo sviluppo di catalizzatori bifunzionali metallo-legante da impiegare in reazioni di catalisi omogenea, in particolare in reazioni redox quali idrogenazione e deidrogenazione attraverso il trasferimento di idrogeno. Il mio progetto ha riguardato la messa a punto della sintesi di complessi di Ru(0) che combinano leganti ciclopentadienonici e carbeni N-eterociclici e la sintesi dei corrispondenti complessi cationici per protonazione. Inoltre, è stato sintetizzato e caratterizzato un nuovo complesso cationico attraverso la metilazione del corrispettivo complesso neutro. I complessi sintetizzati sono stati utilizzati come precursori di catalizzatori nella riduzione tramite trasferimento di idrogeno del 4-fluoroacetofenone, valutandone l’attività catalitica in relazione a leganti, additivi e controioni. Allo scopo di delineare qualche ipotesi sul meccanismo di reazione sono stati effettuati diversi studi sulla reattività dei complessi impiegati in catalisi, in particolare usando la piridina come agente di “trapping”. Infine, è stato condotto uno studio preliminare dell’attività catalitica dei complessi sintetizzati nell’ossidazione di benzilalcol a benzaldeide. The present work is part of a research project that involves the study of new ruthenium-based transition metal complexes in order to develop new metal-ligand bifunctional catalysts to employ in homogeneous catalytic systems, in particular in redox reactions such as hydrogenation and dehydrogenation through hydrogen transfer. My project is focused on the optimization of the synthesis of Ru(0) complexes that combines different ligands as tetraphenylcyclopentadienone and N-heterocyclic carbenes and the synthesis of the corresponding cationic complexes by protonation. Furthermore, it is reported the synthesis and characterization of a new cationic complex obtained by methylation of the corresponding neutral complex. All the prepared complexes were employed as catalyst precursors in the transfer hydrogenation of 4-fluoroacetophenone and their performances were investigated in relation to the type of ligands, additives and counterions. The reactivity of these ruthenium complexes was also investigated with the aim of delineate some hypothesis on the reaction mechanism, in particular employing pyridine as a trapping agent. Finally, preliminary studies on the oxidation of benzyl alcohol have been carried out.

COMBUSTION INITIATION BY ELECTRICAL-DISCHARGE-INDUCED PLASMA IN LEAN AND DILUTE METHANE-AIR MIXTURE: EXPERIMENTAL AND MODELING INVESTIGATION

This dissertation represents experimental and numerical investigations of combustion initiation trigged by electrical-discharge-induced plasma within lean and dilute methane air mixture. This research topic is of interest due to its potential to further promote the understanding and prediction of spark ignition quality in high efficiency gasoline engines, which operate with lean and dilute fuel-air mixture. It is specified in this dissertation that the plasma to flame transition is the key process during the spark ignition event, yet it is also the most complicated and least understood procedure. Therefore the investigation is focused on the overlapped periods when plasma and flame both exists in the system. Experimental study is divided into two parts. Experiments in Part I focuses on the flame kernel resulting from the electrical discharge. A number of external factors are found to affect the growth of the flame kernel, resulting in complex correlations between discharge and flame kernel. Heat loss from the flame kernel to code ambient is found to be a dominant factor that quenches the flame kernel. Another experimental focus is on the plasma channel. Electrical discharges into gases induce intense and highly transient plasma. Detailed observation of the size and contents of the discharge-induced plasma channel is performed. Given the complex correlation and the multi-discipline physical/chemical processes involved in the plasma-flame transition, the modeling principle is taken to reproduce detailed transitions numerically with minimum analytical assumptions. Detailed measurement obtained from experimental work facilitates the more accurate description of initial reaction conditions. The novel and unique spark source considering both energy and species deposition is defined in a justified manner, which is the key feature of this Ignition by Plasma (IBP) model. The results of numerical simulation are intuitive and the potential of numerical simulation to better resolve the complex spark ignition mechanism is presented. Meanwhile, imperfections of the IBP model and numerical simulation have been specified and will address future attentions.

NANOSCALE ELECTROCHEMISTRY BY IN-SITU TRANSMISSION ELECTRON MICROSCOPY

Nanoscale research in energy storage has recently focused on investigating the properties of nanostructures in order to increase energy density, power rate, and capacity. To better understand the intrinsic properties of nanomaterials, a new and advanced in situ system was designed that allows atomic scale observation of materials under external fields. A special holder equipped with a scanning tunneling microscopy (STM) probe inside a transmission electron microscopy (TEM) system was used to perform the in situ studies on mechanical, electrical, and electrochemical properties of nanomaterials. The nanostructures of titanium dioxide (TiO2) nanotubes are characterized by electron imaging, diffraction, and chemical analysis techniques inside TEM. TiO2 nanotube is one of the candidates as anode materials for lithium ion batteries. It is necessary to study their morphological, mechanical, electrical, and electrochemical properties at atomic level. The synthesis of TiO2 nanotubes showed that the aspect ratio of TiO2 could be controlled by processing parameters, such as anodization time and voltage. Ammonium hydroxide (NH4OH) treated TiO2 nanotubes showed unexpected instability. Observation revealed the nanotubes were disintegrated into nanoparticles and the tubular morphology was vanished after annealing. The nitrogen compounds incorporated in surface defects weaken the nanotube and result in the collapse of nanotube into nanoparticles during phase transformation. Next, the electrical and mechanical properties of TiO2 nanotubes were studied by in situ TEM system. Phase transformation of anatase TiO2 nanotubes into rutile nanoparticles was studied by in situ Joule heating. The results showed that single anatase TiO2 nanotubes broke into ultrafine small anatase nanoparticles. On further increasing the bias, the nanoclusters of anatase particles became prone to a solid state reaction and were grown into stable large rutile nanoparticles. The relationship between mechanical and electrical properties of TiO2 nanotubes was also investigated. Initially, both anatase and amorphous TiO2 nanotubes were characterized by using I-V test to demonstrate the semiconductor properties. The observation of mechanical bending on TiO2 nanotubes revealed that the conductivity would increase when bending deformation happened. The defects on the nanotubes created by deformation helped electron transportation to increase the conductivity. Lastly, the electrochemical properties of amorphous TiO2 nanotubes were characterized by in situ TEM system. The direct chemical and imaging evidence of lithium-induced atomic ordering in amorphous TiO2 nanotubes was studied. The results indicated that the lithiation started with the valance reduction of Ti4+ to Ti3+ leading to a LixTiO2 intercalation compound. The continued intercalation of Li ions in TiO2 nanotubes triggered an amorphous to crystalline phase transformation. The crystals were formed as nano islands and identified to be Li2Ti2O4 with cubic structure (a = 8.375 Å). This phase transformation is associated with local inhomogeneities in Li distribution. Based on these observations, a new reaction mechanism is proposed to explain the first cycle lithiation behavior in amorphous TiO2 nanotubes.

Mechanisms of drug-induced allergy

We identified English-language publications on hypersensitivity reactions to xenobiotics through the PubMed database, using the search terms drug and/or xenobiotic, hypersensitivity reaction, mechanism, and immune mediated. We analyzed articles pertaining to the mechanism and the role of T cells. Immune hypersensitivity reactions to drugs are mediated predominantly by IgE antibodies or T cells. The mechanism of IgE-mediated reactions is well investigated, but the mechanisms of T-cell-mediated drug hypersensitivity are not well understood. The literature describes 2 concepts: the hapten/prohapten concept and the concept of pharmacological interactions of drugs with immune receptors. In T-cell-mediated allergic drug reactions, the specificity of the T-cell receptor that is stimulated by the drug may often be directed to a cross-reactive major histocompatibility complex-peptide compound. Thus, previous contact with the causative drug is not obligatory, and an immune mechanism should be considered as the cause of hypersensitivity, even in reactions that occur on primary exposure. Indeed, immune-mediated reactions to xenobiotics in patients without prior exposure to the agent have been described recently for radiocontrast media and neuromuscular blocking agents. Thus, the "allergenic" potential of a drug under development should be evaluated not only by screening its haptenlike characteristics but also by assessing its direct immunostimulatory potential.

Diseño y síntesis de materiales nanoestructurados basados en aluminatos de estroncio con propiedades fotoluminiscentes

Durante la última década, se han llevado acabo numeroso estudios sobre la síntesis de materiales fotoluminiscentes sub-micrónicos, en gran medida, al amplio número de aplicaciones que demandan este tipo de materiales. En concreto dentro de los materiales fosforescentes o también denominados materiales con una prolongada persistencia de la luminiscencia, los estudios se han enfocado en la matriz de SrAl2O4 dopada con Europio (Eu2+) y Disprosio (Dy3+) dado que tiene mayor estabilidad y persistencia de la fosforescencia con respecto a otras matrices. Estos materiales se emplean mayoritariamente en pinturas luminiscentes, tintas, señalización de seguridad pública, cerámicas, relojes, textiles y juguetes fosforescentes. Dado al amplio campo de aplicación de los SrAl2O4:Eu, Dy, se han investigado múltiples rutas de síntesis como la ruta sol-gel, la síntesis hidrotermal, la síntesis por combustión, la síntesis láser y la síntesis en estado sólido con el fin de desarrollar un método eficiente y que sea fácilmente escalable. Sin embargo, en la actualidad el método que se emplea para el procesamiento a nivel industrial de los materiales basados en aluminato de estroncio es la síntesis por estado sólido, que requiere de temperaturas de entre 1300 a 1900oC y largos tiempos de procesamiento. Además el material obtenido tiene un tamaño de partícula de 20 a 100 μm; siendo este tamaño restrictivo para el empleo de este tipo de material en determinadas aplicaciones. Por tanto, el objetivo de este trabajo es el desarrollo de nuevas estrategias que solventen las actuales limitaciones. Dentro de este marco se plantean una serie de objetivos específicos: Estudio de los parámetros que gobiernan los procesos de reducción del tamaño de partícula mediante molienda y su relación en la respuesta fotoluminiscente. Estudio de la síntesis por combustión de SrAl2O4:Eu, Dy, evaluando el efecto de la temperatura y la cantidad de combustible (urea) en el proceso para la obtención de partículas cristalinas minimizando la presencia de fases secundarias. Desarrollo de nuevas rutas de síntesis de SrAl2O4:Eu, Dy empleando el método de sales fundidas. Determinación de los mecanismos de reacción en presencia de la sal fundida en función de los parámetros de proceso que comprende la relación de sales y reactivos, la naturaleza de la alúmina y su tamaño, la temperatura y atmósfera de tratamiento. Mejora de la eficiencia de los procesos de síntesis para obtener productos con propiedades finales óptimas en procesos factibles industrialmente para su transferencia tecnológica. Es este trabajo han sido evaluados los efectos de diferentes procesos de molienda para la reducción del tamaño de partícula del material de SrAl2O4:Eu, Dy comercial. En el proceso de molienda en medio húmedo por atrición se observa la alteración de la estructura cristalina del material debido a la reacción de hidrólisis generada incluso empleando como medio líquido etanol absoluto. Con el fin de solventar las desventajas de la molienda en medio húmedo se llevo a cabo un estudio de la molturación en seco del material. La molturación en seco de alta energía reduce significativamente el tamaño medio de partícula. Sin embargo, procesos de molienda superiores a una duración de 10 minutos ocasionan un aumento del estado de aglomeración de las partículas y disminuyen drásticamente la respuesta fotoluminiscente del material. Por tanto, se lleva a cabo un proceso de molienda en seco de baja energía. Mediante este método se consigue reducir el tamaño medio de partícula, d50=2.8 μm, y se mejora la homogeneidad de la distribución del tamaño de partícula evitando la amorfización del material. A partir de los resultados obtenidos mediante difracción de rayos X y microscopia electrónica de barrido se infiere que la disminución de la intensidad de la fotoluminiscencia después de la molienda en seco de alta energía con respecto al material inicial se debe principalmente a la reducción del tamaño de cristalito. Se observan menores variaciones en la intensidad de la fotoluminiscencia cuando se emplea un método de molienda de baja de energía ya que en estos procesos se preserva el dominio cristalino y se reduce la amorfización significativamente. Estos resultados corroboran que la intensidad de la fotoluminiscencia y la persistencia de la luminiscencia de los materiales de SrAl2O4:Eu2+, Dy3+ dependen extrínsecamente de la morfología de las partículas, del tamaño de partícula, el tamaño de grano, los defectos superficiales e intrínsecamente del tamaño de cristalito. Siendo las características intrínsecas las que dominan con respecto a las extrínsecas y por tanto tienen mayor relevancia en la respuesta fotoluminiscente. Mediante síntesis por combustión se obtuvieron láminas nanoestructuradas de SrAl2O4:Eu, Dy de ≤1 μm de espesor. La cantidad de combustible, urea, en la reacción influye significativamente en la formación de determinadas fases cristalinas. Para la síntesis del material de SrAl2O4:Eu, Dy es necesario incluir un contenido de urea mayor que el estequiométrico (siendo m=1 la relación estequiométrica). La incorporación de un exceso de urea (m>1) requiere de la presencia de un agente oxidante interno, HNO3, para que la reacción tenga lugar. El empleo de un mayor contenido de urea como combustible permite una quelación efectiva de los cationes en el sistema y la creación de las condiciones reductoras para obtener un material de mayor cristalinidad y con mejores propiedades fotoluminiscentes. El material de SrAl2O4:Eu, Dy sintetizado a una temperatura de ignición de 600oC tiene un tamaño medio 5-25 μm con un espesor de ≤1 μm. Mediante procesos de molturación en seco de baja energía es posible disminuir el tamaño medio de partícula ≈2 μm y homogenizar la distribución del tamaño de partícula pero hay un deterioro asociado de la respuesta luminiscente. Sin embargo, se puede mejorar la respuesta fotoluminiscente empleando un tratamiento térmico posterior a 900oC N2-H2 durante 1 hora que no supone un aumento del tamaño de partícula pero si permite aumentar el tamaño de cristalito y la reducción del Eu3+ a Eu2+. Con respecto a la respuesta fotoluminiscente, se obtiene valores de la intensidad de la fotoluminiscencia entre un 35%-21% con respecto a la intensidad de un material comercial de referencia. Además la intensidad inicial del decaimiento de la fosforescencia es un 20% de la intensidad del material de referencia. Por tanto, teniendo en cuenta estos resultados, es necesario explorar otros métodos de síntesis para la obtención de los materiales bajo estudio. Por esta razón, en este trabajo se desarrollo una ruta de síntesis novedosa para sintetizar SrAl2O4:Eu, Dy mediante el método de sales fundidas para la obtención de materiales de gran cristalinidad con tamaños de cristalito del orden nanométrico. Se empleo como sal fundente la mezcla eutéctica de NaCl y KCl, denominada (NaCl-KCl)e. La principal ventaja de la incorporación de la mezcla es el incremento la reactividad del sistema, reduciendo la temperatura de formación del SrAl2O4 y la duración del tratamiento térmico en comparación con la síntesis en estado sólido. La formación del SrAl2O4 es favorecida ya que se aumenta la difusión de los cationes de Sr2+ en el medio líquido. Se emplearon diferentes tipos de Al2O3 para evaluar el papel del tamaño de partícula y su naturaleza en la reacción asistida por sales fundidas y por tanto en la morfología y propiedades del producto final. Se obtuvieron partículas de morfología pseudo-esférica de tamaño ≤0.5 μm al emplear como alúmina precursora partículas sub-micrónicas ( 0.5 μm Al2O3, 0.1 μm Al2 O3 y γ-Al2O3). El mecanismo de reacción que tiene lugar se asocia a procesos de disolución-precipitación que dominan al emplear partículas de alúmina pequeñas y reactivas. Mientras al emplear una alúmina de 6 μm Al2O3 prevalecen los procesos de crecimiento cristalino siguiendo un patrón o plantilla debido a la menor reactividad del sistema. La nucleación y crecimiento de nanocristales de SrAl2O4:Eu, Dy se genera sobre la superficie de la alúmina que actúa como soporte. De esta forma se desarrolla una estructura del tipo coraza-núcleo («core-shell» en inglés) donde la superficie externa está formada por los cristales fosforescentes de SrAl2O4 y el núcleo está formado por alúmina. Las partículas obtenidas tienen una respuesta fotoluminiscente diferente en función de la morfología final obtenida. La optimización de la relación Al2O3/SrO del material de SrAl2O4:Eu, Dy sintetizado a partir de la alúmina de 6 μm permite reducir las fases secundarias y la concentración de dopantes manteniendo la respuesta fotoluminiscente. Comparativamente con un material comercial de SrAl2O4:Eu, Dy de referencia, se han alcanzado valores de la intensidad de la emisión de hasta el 90% y de la intensidad inicial de las curvas de decaimiento de la luminiscencia de un 60% para el material sintetizado por sales fundidas que tiene un tamaño medio ≤ 10μm. Por otra parte, es necesario tener en cuenta que el SrAl2O4 tiene dos polimorfos, la fase monoclínica que es estable a temperaturas inferiores a 650oC y la fase hexagonal, fase de alta temperatura, estable a temperaturas superiores de 650oC. Se ha determinado que fase monoclínica presenta propiedades luminiscentes, sin embargo existen discordancias a cerca de las propiedades luminiscentes de la fase hexagonal. Mediante la síntesis por sales fundidas es posible estabilizar la fase hexagonal empleando como alúmina precursora γ-Al2O3 y un exceso de Al2O3 (Al2O3/SrO:2). La estabilización de la fase hexagonal a temperatura ambiente se produce cuando el tamaño de los cristales de SrAl2O4 es ≤20 nm. Además se observó que la fase hexagonal presenta respuesta fotoluminiscente. El diseño de materiales de SrAl2O4:Eu,Dy nanoestructurados permite modular la morfología del material y por tanto la intensidad de la de la fotoluminiscencia y la persistencia de la luminiscencia. La disminución de los materiales precursores, la temperatura y el tiempo de tratamiento significa la reducción de los costes económicos del material. De ahí la viabilidad de los materiales de SrAl2O4:Eu,Dy obtenidos mediante los procesos de síntesis propuestos en esta memoria de tesis para su posterior escalado industrial. ABSTRACT The synthesis of sub-micron photoluminescent particles has been widely studied during the past decade because of the promising industrial applications of these materials. A large number of matrices has been developed, being SrAl2O4 host doped with europium (Eu2+) and dysprosium (Dy3+) the most extensively studied, because of its better stability and long-lasting luminescence. These functional inorganic materials have a wide field of application in persistent luminous paints, inks and ceramics. Large attention has been paid to the development of an efficient method of preparation of SrAl2O4 powders, including solgel method, hydrothermal synthesis, laser synthesis, combustion synthesis and solid state reaction. Many of these techniques are not compatible with large-scale production and with the principles of sustainability. Moreover, industrial processing of highly crystalline powders usually requires high synthesis temperatures, typically between 1300 a 1900oC, with long processing times, especially for solid state reaction. As a result, the average particle size is typically within the 20-100 μm range. This large particle size is limiting for current applications that demand sub-micron particles. Therefore, the objective of this work is to develop new approaches to overcome these limitations. Within this frame, it is necessary to undertake the following purposes: To study the parameters that govern the particle size reduction by milling and their relation with the photoluminescence properties. To obtain SrAl2O4:Eu, Dy by combustion synthesis, assessing the effect of the temperature and the amount of fuel (urea) to synthesize highly crystalline particles minimizing the presence of secondary phases. To develop new synthesis methods to obtain SrAl2O4:Eu, Dy powders. The molten salt synthesis has been proposed. As the method is a novel route, the reaction mechanism should be determine as a function of the salt mixture, the ratio of the salt, the kind of Al2O3 and their particle size and the temperature and the atmosphere of the thermal treatment. To improve the efficiency of the synthesis process to obtain SrAl2O4:Eu, Dy powders with optimal final properties and easily scalable. On the basis of decreasing the particle size by using commercial product SrAl2O4:Eu2+, Dy3+ as raw material, the effects of different milling methods have been evaluated. Wet milling can significantly alter the structure of the material through hydrolysis reaction even in ethanol media. For overcoming the drawbacks of wet milling, a dry milling-based processes are studied. High energy dry milling process allows a great reduction of the particle size, however milling times above 10 min produce agglomeration and accelerates the decrease of the photoluminescence feature. To solve these issues the low energy dry milling process proposed effectively reduces the particle size to d50=2.8 μm, and improves the homogeneity avoiding the amorphization in comparison with previous methods. The X-ray diffraction and scanning electron microscope characterization allow to infer that the large variations in PL (Photoluminescence) values by high energy milling process are a consequence mainly of the crystallite size reduction. The lesser variation in PL values by low energy milling proces is related to the coherent crystalline domain preservation and the unnoticeable amorphization. These results corroborate that the photoluminescence intensity and the persistent luminescence of the SrAl2O4:Eu2+, Dy3+ powders depend extrinsically on the morphology of the particles such as particle size, grain size, surface damage and intrinsically on the crystallinity (crystallite size); being the intrinsically effects the ones that have a significant influence on the photoluminescent response. By combustion method, nanostructured SrAl2O4:Eu2+, Dy3+ sheets with a thickness ≤1 μm have been obtained. The amount of fuel (urea) in the reaction has an important influence on the phase composition; urea contents larger than the stoichiometric one require the presence of an oxidant agent such as HNO3 to complete the reaction. A higher amount of urea (excess of urea: denoted m>1, being m=1 the stoichiometric composition) including an oxidizing agent produces SrAl2O4:Eu2+,Dy3+ particles with persistent luminescence due to the effective chelation of the cations and the creation of suitable atmospheric conditions to reduce the Eu3+ to Eu2+. Therefore, optimizing the synthesis parameters in combustion synthesis by using a higher amount of urea and an internal oxidizing agent allows to complete the reaction. The amount of secondary phases can be significantly reduced and the photoluminescence response can be enhanced. This situation is attributed to a higher energy that improves the crystallinity of the powders. The powders obtained have a particle size c.a. 5-25 μm with a thickness ≤1 μm and require relatively low ignition temperatures (600oC). It is possible to reduce the particle size by a low energy dry milling but this process implies the decrease of the photoluminescent response. However, a post-thermal treatment in a reducing atmosphere allows the improvement of the properties due to the increment of crystallinity and the reduction of Eu3+ to Eu2+. Compared with the powder resulted from solid state method (commercial reference: average particle size, 20 μm and heterogeneous particle size distribution) the emission intensity of the powder prepared by combustion method achieve the values between 35% to 21% of the reference powder intensity. Moreover, the initial intensity of the decay curve is 20% of the intensity of the reference powder. Taking in account these results, it is necessary to explore other methods to synthesize the powders For that reason, an original synthetic route has been developed in this study: the molten salt assisted process to obtain highly crystalline SrAl2O4 powders with nanometric sized crystallites. The molten salt was composed of a mixture of NaCl and KCl using a 0.5:0.5 molar ratio (eutectic mixture hereafter abbreviated as (NaCl-KCl)e). The main advantages of salt addition is the increase of the reaction rate, the significant reduction of the synthesis temperature and the duration of the thermal treatment in comparison with classic solid state method. The SrAl2O4 formation is promoted due to the high mobility of the Sr2+ cations in the liquid medium. Different kinds of Al2O3 have been employed to evaluate the role of the size and the nature of this precursor on the kinetics of reaction, on the morphology and the final properties of the product. The SrAl2O4:Eu2+, Dy3+ powders have pseudo-spherical morphology and particle size ≤0.5 μm when a sub-micron Al2O3 ( 0.5 μm Al2O3, 0.1 μm Al2O3 and γ-Al2O3) has been used. This can be attributed to a higher reactivity in the system and the dominance of dissolution-precipitation mechanism. However, the use of larger alumina (6 μm Al2O3) modifies the reaction pathway leading to a different reaction evolution. More specifically, the growth of SrAl2O4 sub-micron particles on the surface of hexagonal platelets of 6μm Al2O3 is promoted. The particles retain the shape of the original Al2O3 and this formation process can be attributed to a «core-shell» mechanism. The particles obtained exhibit different photoluminescent response as a function of the final morphology of the powder. Therefore, through this study, it has been elucidated the reaction mechanisms of SrAl2O4 formation assisted by (NaCl-KCl)e that are governed by the diffusion of SrCO3 and the reactivity of the alumina particles. Optimizing the Al2O3/SrO ratio of the SrAl2O4:Eu, Dy powders synthesized with 6 μm Al2O3 as a precursor, the secondary phases and the concentration of dopant needed can be reduced keeping the photoluminescent response of the synthesized powder. Compared with the commercial reference powder, up to 90% of the emission intensity of the reference powder has been achieved for the powder prepared by molten salt method using 6μm Al2O3 as alumina precursor. Concerning the initial intensity of the decay curve, 60% of the initial intensity of the reference powder has been obtained. Additionally, it is necessary to take into account that SrAl2O4 has two polymorphs: monoclinic symmetry that is stable at temperatures below 650oC and hexagonal symmetry that is stable above this temperature. Monoclinic phase shows luminescent properties. However, there is no clear agreement on the emission of the hexagonal structure. By molten salt, it is possible to stabilize the hexagonal phase of SrAl2O4 employing an excess of Al2O3 (Al2O3/SrO: 2) and γ-Al2O3 as a precursor. The existence of nanometric crystalline domains with lower size (≤20 nm) allows the stabilization of the hexagonal phase. Moreover, it has been evidenced that the hexagonal polymorph exhibits photoluminescent response. To sum up, the design of nanostructured SrAl2O4:Eu2+, Dy3+ materials allows to obtain different morphologies and as consequence different photoluminescent responses. The reduction of temperature, duration of the thermal treatment and the precursors materials needed imply the decrease of the economic cost of the material. Therefore, the viability, suitability and scalability of the synthesis strategy developed in this work to process SrAl2O4:Eu2+, Dy3+ are demonstrated.

Identification of 5′-deoxyribose phosphate lyase activity in human DNA polymerase γ and its role in mitochondrial base excision repair in vitro

Mitochondria have been proposed to possess base excision repair processes to correct oxidative damage to the mitochondrial genome. As the only DNA polymerase (pol) present in mitochondria, pol γ is necessarily implicated in such processes. Therefore, we tested the ability of the catalytic subunit of human pol γ to participate in uracil-provoked base excision repair reconstituted in vitro with purified components. Subsequent to actions of uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease, human pol γ was able to fill a single nucleotide gap in the presence of a 5′ terminal deoxyribose phosphate (dRP) flap. We report here that the catalytic subunit of human pol γ catalyzes release of the dRP residue from incised apurinic/apyrimidinic sites to produce a substrate for DNA ligase. The heat sensitivity of this activity suggests the dRP lyase function requires a three-dimensional protein structure. The dRP lyase activity does not require divalent metal ions, and the ability to trap covalent enzyme-DNA complexes with NaBH4 strongly implicates a Schiff base intermediate in a β-elimination reaction mechanism.

Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a

Chlorophyll b is an ubiquitous accessory pigment in land plants, green algae, and prochlorophytes. Its biosynthesis plays a key role in the adaptation to various light environments. We isolated six chlorophyll b-less mutants by insertional mutagenesis by using the nitrate reductase or argininosuccinate lyase genes as tags and examined the rearrangement of mutant genomes. We found that an overlapping region of a nuclear genome was deleted in all mutants and that this encodes a protein whose sequence is similar to those of methyl monooxygenases. This coding sequence also contains putative binding domains for a [2Fe-2S] Rieske center and for a mononuclear iron. The results demonstrate that a chlorophyll a oxygenase is involved in chlorophyll b formation. The reaction mechanism of chlorophyll b formation is discussed.

No ·NO from NO synthase

The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of l-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form l-citrulline (Cit) and nitric oxide (·NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify ·NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable ·NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal ·NO standards were only partially recovered and the dismutation of superoxide (O2⨪), which otherwise scavenges ·NO to yield ONOO−, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO− resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8–1.1, suggesting either that additional reducing equivalents besides NADPH are required to explain Arg oxidation to ·NO or that ·NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H4Bip. Here, recovery of internal ·NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO−) to ·NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from ·NO decomposition, was consistent with formation of an ·NO precursor molecule such as NO−. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that ·NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.

Substitutions in the aspartate transcarbamoylase domain of hamster CAD disrupt oligomeric structure

Aspartate transcarbamoylase (ATCase; EC 2.1.3.2) is one of three enzymatic domains of CAD, a protein whose native structure is usually a hexamer of identical subunits. Alanine substitutions for the ATCase residues Asp-90 and Arg-269 were generated in a bicistronic vector that encodes a 6-histidine-tagged hamster CAD. Stably transfected mammalian cells expressing high levels of CAD were easily isolated and CAD purification was simplified over previous procedures. The substitutions reduce the ATCase Vmax of the altered CADs by 11-fold and 46-fold, respectively, as well as affect the enzyme's affinity for aspartate. At 25 mM Mg2+, these substitutions cause the oligomeric CAD to dissociate into monomers. Under the same dissociating conditions, incubating the altered CAD with the ATCase substrate carbamoyl phosphate or the bisubstrate analogue N-phosphonacetyl-l-aspartate unexpectedly leads to the reformation of hexamers. Incubation with the other ATCase substrate, aspartate, has no effect. These results demonstrate that the ATCase domain is central to hexamer formation in CAD and suggest that the ATCase reaction mechanism is ordered in the same manner as the Escherichia coli ATCase. Finally, the data indicate that the binding of carbamoyl phosphate induces conformational changes that enhance the interaction of CAD subunits.

Covalent intermediate trapped in 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase structure at 1.95-Å resolution

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. The enzyme is a class I aldolase whose reaction mechanism involves formation of Schiff base intermediates between Lys-133 and a keto substrate. A covalent adduct was trapped by flash freezing KDPG aldolase crystals soaked with 10 mM pyruvate in acidic conditions at pH 4.6. Structure determination to 1.95-Å resolution showed that pyruvate had undergone nucleophilic attack with Lys-133, forming a protonated carbinolamine intermediate, a functional Schiff base precursor, which was stabilized by hydrogen bonding with active site residues. Carbinolamine interaction with Glu-45 indicates general base catalysis of several rate steps. Stereospecific addition is ensured by aromatic interaction of Phe-135 with the pyruvate methyl group. In the native structure, Lys-133 donates all of its hydrogen bonds, indicating the presence of an ɛ-ammonium salt group. Nucleophilic activation is postulated to occur by proton transfer in the monoprotonated zwitterionic pair (Glu-45/Lys-133). Formation of the zwitterionic pair requires prior side chain rearrangement by protonated Lys-133 to displace a water molecule, hydrogen bonded to the zwitterionic residues.