La acetilcolina ejerce un rol preponderante como neuromodulador en el sistema nervioso central, principalmente a nivel de los circuitos neuronales asociados a cognición, memoria, aprendizaje, atención, recompensa y procesamiento de la información sensorial, modificando su respuesta frente a estímulos internos y externos. La señalización colinérgica ocurre tanto por mecanismos de transmisión sináptica como extrasináptica, siendo estos últimos los principales involucrados en la modulación de la excitabilidad neuronal, la liberación presináptica de neurotransmisores, la plasticidad neuronal y la activación coordinada de distintos grupos de neuronas en el encéfalo. El efecto neuromodulador de la acetilcolina depende no sólo de su sitio de liberación y de la población neuronal blanco, sino también de los receptores a través de los cuales actúa. Por lo tanto, los receptores nicotínicos ionotrópicos constituyen una pieza clave en el sistema colinérgico del encéfalo. Estos son canales iónicos pentaméricos activados por ligando que forman parte de la familia de receptores Cys-loop. Son permeables a cationes y pueden estar formados por cinco subunidades idénticas o por combinaciones de subunidades diferentes, dando lugar a receptores homoméricos o heteroméricos respectivamente. Cada subtipo de receptor nicotínico presenta una estequiometría definida, es decir, una composición y un arreglo espacial de subunidades particular, responsable de las propiedades biofísicas, farmacológicas y funcionales del canal, que se encuentran íntimamente asociadas a su rol fisiológico diferencial. Una de las subunidades de receptor nicotínico más abundantes en el sistema nervioso central es α7, tradicionalmente reconocida por su capacidad para formar receptores homoméricos de origen evolutivo más ancestral. Sin embargo, evidencias recientes demostraron que también posee la habilidad de coensamblarse con otras subunidades para generar receptores heteroméricos, tales como el novedoso subtipo α7β2. Numerosos desórdenes neurológicos, psiquiátricos, inflamatorios y neurodegenerativos se presentan acompañados de una deficiente señalización colinérgica, por lo que estos receptores nicotínicos conformados por subunidades α7 constituyen un promisorio blanco terapéutico. En el presente trabajo de tesis se descifraron las bases moleculares de la activación y modulación de los receptores nicotínicos α7 homoméricos y heteroméricos, utilizando principalmente técnicas electrofisiológicas, con el fin de comprender el impacto diferencial de ambos subtipos en la señalización colinérgica del sistema nervioso central. El capítulo I fue dividido en tres partes y se centró en el estudio de la función molecular del receptor α7 homomérico a nivel macroscópico y microscópico. Este receptor exhibe propiedades cinéticas y farmacológicas particulares que lo tornan óptimo en la mediación de procesos de transmisión extrasinápticos, incluyendo su elevada permeabilidad a los iones calcio y su capacidad para transformar respuestas ionotrópicas transitorias en eventos de señalización metabotrópica sostenidos. En la parte I se analizaron los mecanismos moleculares de activación y modulación del receptor α7 homomérico. En presencia de acetilcolina el receptor se activó y desensibilizó con una cinética rápida, evocando mayoritariamente aperturas aisladas breves. Por el contrario, la activación alostérica presentó un mecanismo molecular radicalmente diferente, con un enlentecimiento de la cinética de desensibilización, que permitió la prolongación de las aperturas individuales y su agrupación en episodios de activación sostenidos de muy larga duración. Asimismo, se evaluó la potenciación por compuestos prototipo previamente reportados como moduladores alostéricos positivos (PAMs), clasificados como tipo I o II en base a sus efectos a nivel de corrientes macroscópicas. Si bien ambos incrementaron el pico de la corriente, los PAMs tipo I produjeron mínimos o nulos cambios en la velocidad de desensibilización del receptor, mientras que los PAMs tipo II disminuyeron dicha velocidad notoriamente e incrementaron la reactivación de receptores desde el estado desensibilizado. Para descifrar el origen molecular de dichos efectos se exploró la potenciación a nivel de corrientes unitarias. Se demostró que los PAMs no afectaron la amplitud máxima del canal, incrementaron el tiempo de estado abierto e indujeron la aparición de episodios de activación sostenidos, modificando así la cinética del receptor en los dos casos. Mientras que los PAMs tipo I promovieron episodios de activación con una duración moderada denominados bursts, los PAMs tipo II generaron clusters de muy larga duración, conformados por varios bursts agrupados. La potenciación por ambos tipos de PAMs también fue diferencialmente afectada por la temperatura, siendo los PAMs tipo II los más sensibles, con una actividad potenciadora significativamente menor a temperatura fisiológica en comparación con el accionar a temperatura ambiente. Esta información resulta muy relevante dado que usualmente la caracterización de los PAMs in vitro es llevada a cabo a temperatura ambiente, pero para su potencial uso terapéutico es indispensable el conocimiento de sus efectos a temperaturas fisiológicas. Además, se comprobó que los determinantes estructurales transmembrana influyen en la potenciación por los dos tipos de PAMs, principalmente en los PAMs tipo II, mientras que los PAMs tipo I resultan menos sensibles. Sin embargo, también se identificaron algunos PAMs tipo I notablemente influenciados por los determinantes transmembrana, con un comportamiento intermedio, más semejante al de los PAMs tipo II. En conjunto, estos resultados aportan información esencial para la implementación de agonistas y PAMs de α7 como agentes terapéuticos en desórdenes donde exista una señalización colinérgica deficiente. En la parte II se reveló una conexión entre los efectos benéficos comunes ejercidos por los flavonoides y el receptor α7 homomérico en el sistema nervioso central, ya que estos compuestos naturales presentaron actividad α7-PAM. Se evaluaron prototipos de las tres principales clases de flavonoides: genisteína, quercetina y 5,7-dihidroxi-4-fenilcumarina como neoflavonoide. Se demostró que todos ellos potenciaron las corrientes macroscópicas y microscópicas evocadas por la acetilcolina y no recuperaron receptores desde el estado desensibilizado. Dicha caracterización funcional permitió clasificarlos como PAMs tipo I. Utilizando receptores α7 mutantes y quiméricos se comprobó que los flavonoides comparten determinantes estructurales de potenciación transmembrana con otros PAMs. Estos compuestos polifenólicos no sólo aumentaron la respuesta ionotrópica del receptor α7, sino también la metabotrópica. Se verificó que los mismos potenciaron la disminución de la generación de especies reactivas de oxígeno intracelulares ejercida por la activación de α7, en forma independiente de su efecto antioxidante intrínseco. Tanto es así que la actividad α7-PAM de los flavonoides se propone como un mecanismo adicional por el cual estos compuestos naturales ejercerían su acción antioxidante y su efecto neuroprotector mediado por receptor. En la parte III se sintetizó una novedosa serie de compuestos sintéticos derivados de 1,2,3-triazol 1,4-disustituidos con grupos arilo y fosfonato, que presentaron actividad α7-PAM. El compuesto de mayor eficacia fue el derivado funcionalizado con el grupo fosfonato de metilo, el cual se comportó como PAM tipo I, potenciando las corrientes macroscópicas y microscópicas evocadas por la acetilcolina de forma compatible con su clasificación. La potenciación por este compuesto fue influenciada por los determinantes estructurales transmembrana, compartidos también por los PAMs tipo II. Además, se aplicaron diversas estrategias de relación estructura-actividad para obtener derivados más eficaces, como modificaciones en la longitud de las cadenas carbonadas, variación del grupo aromático e inversión de la geometría del triazol. Esta última fue la única que permitió conservar el accionar α7-PAM del derivado con fosfonato de metilo. Por lo tanto, estos hallazgos permiten proponer a este compuesto como un novedoso farmacóforo de interés, que no sólo exhibe actividad potenciadora por sí mismo, sino que también resulta útil como plataforma para el desarrollo de nuevos agentes potencialmente terapéuticos. El capítulo II se centró en el estudio de la función molecular del receptor α7 heteromérico, particularmente del receptor α7β2, cuya presencia ha sido confirmada en cerebro anterior basal, corteza e hipocampo. Para descifrar el impacto de la incorporación de subunidades β2 en el pentámero de subunidades α7, se generaron receptores con estequiometrías definidas y controladas mediante dos estrategias: la tecnología de concatámeros y la técnica de electrical fingerprinting, que utilizan subunidades covalentemente unidas y no enlazadas respectivamente. Se determinó que las estequiometrías conteniendo una, dos y tres subunidades β2 dieron lugar a receptores α7β2 funcionales. En relación a la activación ortostérica, a medida que el número de subunidades β2 aumentó en el arreglo pentamérico, la amplitud y la conductancia unitaria permanecieron constantes, mientras que la duración de las aperturas y de los episodios de activación sostenidos se prolongaron significativamente, debido a una disminución en la velocidad de desensibilización. La activación en episodios sostenidos o bursts constituyó la marca o huella cinética distintiva del receptor heteromérico α7β2 con respecto al receptor α7 homomérico. Además, la subunidad β2 no aportó su cara complementaria al sitio ortostérico de unión, sino que la activación eficaz por acetilcolina ocurrió únicamente a través de interfaces α7/α7. En relación a los PAMs, originalmente desarrollados como selectivos para el receptor α7 homomérico, se observó que también ejercieron modulación alostérica positiva sobre el receptor α7β2 heteromérico, pero de forma diferencial. Los PAMs tipo I fueron más selectivos para el receptor homomérico, ya que su actividad potenciadora disminuyó con la presencia de subunidades β2 en el pentámero hasta ser prácticamente nula. En cambio, los PAMs tipo II no resultaron selectivos, dado que potenciaron por igual a los receptores α7 homoméricos y heteroméricos, enmascarando sus diferencias cinéticas intrínsecas. Lo mismo ocurrió con la activación alostérica por agonistas alostéricos, que además poseen la capacidad de actuar como PAMs tipo II. Por lo tanto, este estudio constituye la primera caracterización a nivel de canal único de receptores α7 heteroméricos, demostrando las posibles estequiometrías que dan origen a receptores funcionales y la huella cinética de cada uno. Estos resultados contribuyen al entendimiento del rol distintivo de este nuevo receptor y a su identificación funcional en sistemas nativos, ya que por el momento no existen ligandos selectivos. Además, dado que los PAMs de α7 también potenciaron a α7β2, se propone a la selectividad diferencial como un parámetro importante a considerar para el diseño y desarrollo de moduladores más específicos. También se generaron receptores heteroméricos α7β4 con estequiometrías definidas, para comparar el impacto diferencial de la presencia de distintas subunidades β en el arreglo pentamérico. Las estequiometrías conteniendo una y dos subunidades β4 dieron lugar a receptores α7β4 funcionales. En la activación ortostérica de los mismos por acetilcolina, se observó que a mayor número de subunidades β4 en el pentámero la amplitud de las corrientes unitarias aumenta, a diferencia de lo ocurrido con β2; mientras que las duraciones de las aperturas y de los episodios de activación se incrementan de forma similar a lo ocurrido con β2, pero más notoriamente, con una alta frecuencia. De este modo, la funcionalidad de los receptores α7β4 en el sistema de expresión heterólogo empleado, junto con otras evidencias experimentales, sustentan la potencial existencia de otros receptores α7 heteroméricos in vivo, además del subtipo α7β2 ya identificado en el sistema nervioso central.En conjunto, el presente trabajo de tesis permitió descifrar los mecanismos moleculares de activación y modulación alostérica de los receptores homoméricos α7 y heteroméricos α7β2, los cuales exhibieron propiedades y perfiles funcionales particulares. Esto sugiere que ambos subtipos de receptores α7 podrían actuar in vivo a distinta escala temporal, pero con un patrón espacial compartido en algunas áreas del sistema de neuromodulación colinérgico, ejerciendo un impacto diferencial en los circuitos neuronales asociados a cognición y memoria. La información aportada contribuye entonces no sólo a la dilucidación del rol fisiológico distintivo de los receptores α7 homoméricos y heteroméricos con sus respectivas implicancias fisiopatológicas, sino también al diseño, desarrollo y optimización de nuevos agentes terapéuticos más selectivos. / Acetylcholine plays a major role as neuromodulator in the central nervous system, mainly at the level of neural circuits associated to cognition, memory, learning, attention, reward and sensory gating, by modifying the response to internal and external inputs. Cholinergic signaling occurs by synaptic and extrasynaptic transmission, the latter being the main mechanism involved in the modulation of neuronal excitability, presynaptic neurotransmitter release, neuronal plasticity and coordinated firing of groups of neurons in the brain. The effect of acetylcholine as a neuromodulator depends not only on the site of release and the target neuron, but also on the receptors through which acetylcholine acts. In this regard, the ionotropic nicotinic receptors are a key piece in the brain cholinergic system. They are pentameric ligand-gated ion channels, members of the Cys-loop receptor family. Nicotinic receptors are permeable to cations and can be formed by five identical subunits or different subunit combinations, giving place to homomeric or heteromeric receptors, respectively. Each nicotinic receptor subtype has a defined stoichiometry, which means a particular composition and spatial arrangement of subunits, that is responsible for the biophysical, pharmacological and functional properties of the channel and it is intimately related to the differential physiological role. One of the most abundant nicotinic subunits in the central nervous system is α7, traditionally known by its ability to form homomeric receptors due to its ancestral evolutionary origin. However, recent evidence demonstrated that α7 has also the ability to co-assemble with other subunits to generate heteromeric receptors, for instance the novel α7β2 subtype. Several neurological, mental, inflammatory and neurodegenerative disorders are accompanied by a defective cholinergic signaling, therefore, α7 nicotinic receptors are promising therapeutic targets. In this thesis, by using electrophysiological techniques, we deciphered the molecular basis of the activation and modulation of α7 homomeric and heteromeric nicotinic receptors, with the final aim of elucidating the differential impact of both subtypes in the cholinergic signaling in the central nervous system. Chapter I was divided into three parts and was focused on the study of the molecular function of α7 homomeric receptor at the macroscopic and microscopic level. This receptor exhibits particular kinetic and pharmacological properties that make it ideal for mediating extrasynaptic transmission processes, including high permeability to calcium ions and the ability to transform transient ionotropic responses into sustained metabotropic signaling events. In Part I, the molecular mechanisms of activation and modulation of α7 homomeric receptor were analysed. In the presence of acetylcholine, the receptor activated and desensitized with rapid kinetics, mostly evoking brief isolated openings. On the contrary, allosteric activation showed a radically different molecular mechanism, with a slowdown in the desensitization kinetics that allows the prolongation of individual openings and their grouping into very long-lasting sustained activation episodes. We also evaluated the potentiation by prototype compounds, previously reported as positive allosteric modulators (PAMs) and classified as type I or II depending on their effect at the macroscopic current level. Although both types of PAMs increased the peak current, type I PAMs produced minimal or no changes in the receptor desensitization rate, whereas type II PAMs decreased notoriously that rate and increased reactivation of receptors from desensitized states. To decipher the molecular origin of these effects, potentiation was explored at the single-channel current level, and revealed that both type of PAMs did not affect the maximal channel amplitude, increased open-channel lifetime and induced sustained activation episodes, thus modifying receptor kinetics in both cases. While type I PAMs provoked activation episodes with moderate duration named bursts, type II PAMs generated very long duration clusters, consisting of several grouped bursts. We showed that potentiation by both types of PAMs was differentially affected by temperature, being type II PAMs more sensitive and less active at a more physiological temperature than at room temperature. This information is useful given that PAMs have been usually characterized in vitro at room temperature, but for their therapeutic use it is necessary to know their effects at more physiological temperatures. In addition, it was demonstrated that transmembrane structural determinants influence potentiation by both types of PAMs, mainly by type II PAMs, while type I PAMs are less sensitive. Nevertheless, some type I PAMs significantly influenced by transmembrane determinants were also identified, with an intermediate behaviour, more similar to that of type II PAMs. Overall, these results provide important information required for the implementation of α7 agonists and PAMs as therapeutic tools for disorders with a defective cholinergic signaling. In Part II, a link between the common beneficial effects exerted by flavonoids and α7 homomeric receptor in the central nervous system was revealed, since these natural compounds showed α7-PAM activity. We evaluated prototypes from the three main flavonoids classes: genistein, quercetin and 5,7-dihydroxy-4-phenylcoumarin as a neoflavonoid. All flavonoids enhanced macroscopic and microscopic currents elicited by acetylcholine and did not recover receptors from the desensitized state, thus allowing us to classify them as type I PAMs. By using mutant and chimeric α7 receptors, we demonstrated that flavonoids share transmembrane structural determinants with other PAMs. These polyphenolic compounds not only increased the receptor ionotropic response, but also the metabotropic one, further reducing the generation of intracellular reactive oxygen species elicited by α7 activation, independently of their direct antioxidant activity. Thus, we propose α7-PAM activity of flavonoids as an additional mechanism by which these natural compounds might exert their receptor-mediated antioxidant action and neuroprotective effect. In Part III, a novel series of synthetic compounds derived from 1,2,3-triazoles 1,4-disubstituted with aryl and phosphonate groups that exhibited α7-PAM activity was synthetized. The most effective compound was the methyl phosphonate-functionalized derivative, which behaves as type I PAM, enhancing the macroscopic and microscopic currents elicited by acetylcholine, in a manner compatible with its classification. Potentiation was influenced by transmembrane structural determinants, also shared by type II PAMs. In addition, several structure-activity relationship strategies were applied to obtain more effective derivatives, for instance modifications of the carbon chains lengths, variation of the aromatic group and inversion of the triazole geometry. The last one allowed the compound to preserve the α7-PAM activity of the methyl phosphonate derivative. Therefore, these results propose this compound not only as a novel pharmacophore with intrinsic PAM activity, but also as a scaffold for the development of new potential therapeutic agents. Chapter II was focused on the study of the molecular function of α7 heteromeric receptors, particularly α7β2 receptor, whose presence has been confirmed in basal forebrain, cortex and hippocampus. To decipher the impact of β2 subunits incorporation into the pentamer of α7 subunits, receptors with fixed and controlled stoichiometries were generated by two approaches: concatemeric technology and electrical fingerprinting technique, which use concatenated and unlinked subunits respectively. It was determined that stoichiometries containing one, two and three β2 subunits gave place to functional α7β2 receptors. Regarding orthosteric activation, as the number of β2 subunits increased in the pentameric arrangement, the amplitude and the single-channel conductance remained constant, while durations of openings and sustained activation episodes were significantly prolonged, due to a decrease in the desensitization rate. Activation in sustained episodes or bursts conforms the kinetic signature of α7β2 heteromeric receptors with respect to α7 homomeric receptor. Moreover, β2 subunit did not provide the complementary face of the orthosteric binding site, so the efficacious activation by acetylcholine occurred only through α7/α7 interfaces. Originally developed as selective for α7 homomeric receptors, we showed that PAMs also exerted positive allosteric modulation on α7β2 heteromeric receptors, but in a differential manner. On the one hand, type I PAMs were more selective for the homomeric receptor, since their potentiating activity decreased with the presence of β2 subunits in the pentamer up to be practically null. On the other hand, type II PAMs were not selective, because they enhanced equally all α7 homomeric and heteromeric receptors, masking their intrinsic kinetic differences. The same occurred with the allosteric activation by allosteric agonists, which also have the capacity to act as type II PAMs. This study constitutes the first single-channel characterization of α7 heteromeric receptors, revealing the stoichiometries that may result in functional receptors and the kinetic signature of each one. These results contribute to the understanding of the distinctive role of this novel receptor and will help to its functional identification in native systems because no selective ligands are available to date. Since α7 PAMs also potentiated α7β2 receptors, we propose the differential selectivity as an important parameter to consider for the design and development of more specific modulators. Furthermore, α7β4 heteromeric receptors with fixed stoichiometry were generated in order to compare the differential impact of distinct β subunits presence into the pentameric arrangement. Stoichiometries containing one and two β4 subunits gave place to functional α7β4 receptors. Regarding orthosteric activation by acetylcholine, it was observed that as the number of β4 subunits in the pentamer increases, the single-channel amplitude rises unlike what occurred with β2, whereas durations of openings and sustained activation episodes increase similarly to what occurred with β2 subunits, but more significantly, with a higher frequency. The functionality of α7β4 receptors in the heterologous expression system used, together with other experimental evidences, support the potential existence of other α7 heteromeric receptors in vivo, besides the α7β2 subtype already identified in central nervous system. On the whole, this thesis work allowed to decipher the molecular mechanisms of activation and allosteric modulation of α7 homomeric and α7β2 heteromeric receptors, which exhibited particular functional profiles and properties. It suggests that both α7 receptors subtypes could act in vivo at distinct temporal scale, but with a shared spatial pattern in some areas of the neuromodulation cholinergic system, exerting a differential impact at the neural circuits associated to cognition and memory. The provided information not only contributes to elucidating the physiological role of α7 homomeric and heteromeric receptors with their respective pathophysiological implications, but also to the design, development and optimization of novel more selective therapeutic agents.

Made available in DSpace on 2014-06-11T19:30:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-06-30Bitstream added on 2014-06-13T19:06:17Z : No. of bitstreams: 1 ricardi_es_me_sjrp.pdf: 2273724 bytes, checksum: eed74e01870d5b90b7fb020a587b9ac0 (MD5) / A Glutationa (GSH) é um tripeptídeo presente nas células, que em sua forma reduzida age como antioxidante, mantendo os grupamentos tiólicos nas proteínas em estado reduzido. Ao exercer função protetora, a glutationa é oxidada para sua forma dissulfeto, expondo quatro cargas negativas. A glutationa oxidada (GSSG) poderia interagir não covalentemente com moléculas capazes de ligar ânions, como a hemoglobina. Essa condição nos levou a investigar as possíveis mudanças estruturais e funcionais das hemoglobinas bovina, humana e majoritária do peixe matrinxã (Brycon cephalus) quando interagem alostericamente com a glutationa. As amostras de sangue bovina e humana foram purificadas e submetidas à eletroforese para verificação de pureza. As propriedades de ligação com oxigênio e controle alostérico foram analisados por tonometria a 20°C, calculando a afinidade de ligação com o O2 e a cooperatividade (n50). As condições experimentais adotadas foram, stripped, cloreto e glutationa oxidada (GSSG), nessas condições, as hemoglobinas bovina e humana apresentaram efeito Bohr alcalino. O cloreto induziu o maior efeito de diminuição da afinidade em praticamente todas as condições. As hemoglobinas apresentaram processo cooperativo de ligação de oxigênio, em todas as condições e em toda faixa de pH. No caso da hemoglobina bovina adulta, a GSSG aumentou a afinidade por O2, em toda a faixa de pH testada, enquanto para a humana isso ocorreu abaixo de pH 7,0. O cloreto capaz de diminuir a afinidade de forma efetiva em todas as condições. A hemoglobina bovina apresentou uma menor afinidade de ligação ao oxigênio em relação à hemoglobina humana. A glutationa oxidada atuou como efetor alostérico heterotrópico em hemoglobinas humana, bovina e matrinxã (Hb-II), aumentando a afinidade de ligação por oxigênio da hemoglobina. Entretanto, a glutationa na forma reduzida... / Glutathione (GSH) is an intracellular tripeptide, which in its reduced form acts as an antioxidant, keeping the cysteines in the reduced state. When exerting protective function, glutathione is oxidized to form its disulfide, exposing four negative charges. The oxidized glutathione (GSSG) could interact with molecules covalently able to bind anions, such as hemoglobin. That motivated us to investigate the possible structural and functional changes of bovine, human and a fish hemoglobin from the fish ‘matrinxã’ (Brycon cephalus) when they interact allosterically with glutathione. Samples of human and cattle blood were purified and subject to electrophoresis to verify their purity. The functional properties of oxygen binding and allosteric control were analyzed by the tonometric method at 20°C, calculating the affinity of O2-binding and cooperativity (n50). The tested experimental conditions involved hemoglobin in the absence (stripped) and in the presence of chloride and oxidized glutathione (GSSG). Under those circumstances, the human and bovine hemoglobins displayed an alkaline Bohr effect. Chloride induced the greatest effect of reducing affinity in all conditions. For all the experimental sets oxygen binding was cooperative. For bovine adult hemoglobin GSSG increased O2-affinity for all the tested pH values, whereas it occurred for human Hb for pH values above 7.0. Bovine Hb displayed lower O2-affinity than its human counterpart. Oxidized glutahione acted as a heterotropic allosteric effector in all the tested hemoglobins, increasing O2-affinity. However, GSH decreased O2-affinity of human and bovine Hbs. We assume that there is a binding site for GSSG at the R state, increasing O2-affinity, and another one for GSH at the T state, stabilizing its conformation and therefore lowering O2-affinity.

A enzima Glucosamina-6-fosfato desaminase (GlcN6P desaminase) é envolvida na conversão reversível da D-glucosamina-6-fosfato (GlcN6P) em Fru6P e amônia, como parte do caminho metabólico de aminoaçúcares como fonte de energia celular. A enzima hexamérica (peso mol. 178200) exibe uma cooperatividade homotrópica intensa em direção à GlcN6P a qual é modulada alostericamente pelo ativador N-acetil-D-glucosamina 6-fosfato (GlcNAc6P). A GlcN6P desaminase foi cristalizada no grupo espacial R32, com parâmetros de rede a = b = 125.9 &#197 e c = 223.2 &#197 e um conjunto de dados à 2.1 &#197 de resolução foi coletado usando radiação de luz síncrotron (Horjales et ai., 1992). A procura no banco de dados de seqüências OWL não mostrou homologia significante com qualquer outra família de proteína, desta maneira a determinação da estrutura foi feita pela técnica de substituição isomórfica múltipla (MIR) a partir de dois derivados, um composto de platina, o K2PtCl4 e um complexo de mercúrio, o ácido mersálico. O mapa MIR a 3 &#197 de resolução mostrou contornos claros e utilizando técnicas de nivelamento de solvente (solvent flattening) estendeu-se as fases até 2.5 &#197. A enzima cristaliza-se com dois monômeros na unidade assimétrica. A densidade eletrônica final foi interpretada com o auxílio do programa gráfico \'O\', sendo possível determinar sem ambigüidade 230 dos 266 resíduos de cada monômero; a partir daí foram usados subseqüentes mapas de Fourier diferença para a localização de todos os outros resíduos. O refinamento do modelo foi feito utilizando o programa X-PLOR (Brünger, 1993), usando a rotina simulated annealing, obtendo o fator R final de 17.4% com 348 moléculas de água e quatro íons inorgânicos de fosfato. O enovelamento do monômero tem uma estrutura do tipo &#945/&#946 com uma folha-&#946 pregueada paralela central com sete fitas com topologia 4x, 1x, 1x, -3x, -1x, -1x, envolvida por ambos os lados por oito hélices-&#945 e uma hélice 310 com duas voltas. A sexta fita da folha-&#946 central tem um prolongamento no C-terminal que faz parte de uma segunda folha-&#946 antiparalela de três fitas com topologia 2, -1. O hexâmero tem uma simetria local 32, com dois trímeros empacotados frente-a-frente com uma rotação relativa de 15&#176 em tomo do eixo de ordem 3 e ligados por pontes salinas e algumas interações hidrofóbicas em tomo do eixo não cristalográfico de ordem 2. As moléculas de cada trímero formam um contato não usual de três resíduos Cis 219 próximo ao eixo de ordem três. Os complexos com ativador alostérico (GlcNAc6P) e inibidor competitivo (2-desoxi 2-amino glucitol 6-fosfato) foram co-cristalizados isomorficamente com a estrutura nativa. Os mapas Fourier diferença mostram claramente densidades para os ligantes, definindo sem ambigüidade o sítio ativo e alostérico. O refinamento dos complexos produziu a mesma conformação da proteína nativa, na margem de erro experimental. Os sítios alostéricos (seis) estão localizados na interface adjacente dos monômeros de cada trímero e os sítios ativos (ou catalíticos) no lado externo de cada monômero, no C-terminal da folha-&#946 central. O monômero tem uma topologia com enovelamento similar a um domínio de ligação de NAD, excluindo os segmentos de aminoácidos 1-35, 145-188 e 243-266. As estruturas dos complexos e da nativa estão em um estado alostérico R em concordância com o modelo MWC para um sistema do tipo K (Monod et al, 1965). Um mecanismo alostérico similar ao da GlcN6P desaminase é encontrado na enzima fosfofrutoquinase (Evans, 1981). Um mecanismo catalítico é proposto para a reação de isomerisação-desaminação da enzima GlcN6P desaminase a partir do mecanismo geral para aldose-cetona isomerases. / The enzyme Glucosamine-6-phosphate deaminase (GlcN6P deaminase) is involved in the reversible conversion of D-glucosamine-6-phosphate (GlcN6P) into Fru6P and ammonia. The hexameric enzyme (mol.wt.=178200) exhibits an intense homotropic co-operativity towards GlcN6P which is allosterically modulated by the activator N-acetyl-D-glucosamine 6-phosphate (GlcNAc6P). The GlcN6P deaminase was crystallized in space group R32, with cell parameters a=b= 125.9 &#197 and c = 223.2 &#197 and a native dataset was collected to 2.1 &#197 resolution at a synchrotron source (Horjales et al, 1992). A search of the OWL sequences database has shown no significant homology with any other known protein family. Therefore, the structure determination will have to be achieved through the Multiple Isomorphous Replacement technique from two isomorphous derivatives, a platinum compound K2PtCl4 and a mercury complex, mersalyl acid. The MIR map at 3 &#197 resolution showed clear molecular boundaries and solvent flattening techniques (Wang, 1985) were used to extend the phase set to 2.5 &#197. The final electron density map was interpreted with the aid of the graphic program \'O\'. The enzyme crystallizes with a dimmer in the asymmetric unit and 230 out of the total 266 residues of each crystallographically independent monomer could be unambiguously identified in the map. The remaining residues were located after subsequent difference Fourier maps. The refinement was made with program X-PLOR (Brunger, 1993), using the simulated annealing routine, obtained R=17.4 % with 348 water molecules and four inorganic phosphate ions. The monomer fold shows an &#945/&#946 structure with a central 7-stranded &#946-sheet with topology 4x, 1x, 1x, -3x, -1x, -1x, surrounded on both sides by eight &#945-helices and 2-turn 310 -helix. The sixth strand of the central &#946-sheet is common to a second 3-stranded anti-parallel &#946-sheet with topology 2, -1. The hexamer has local 32 symmetry, with two trimmers packed in a face-to-face arrangement with a relative rotation of 15&#176 around the 3-fold axis, and linked together by salt-bridge and some hydrophobic contacts. The molecules of each trimmer have extensive contacts and show an unusual feature of the three Cys219 residues closely clustered around the 3-fold axis. The complexes with allosteric activator (GlcNAc6P) and inhibitor (2-deoxy-2-amino glucitol 6-phosphate) were co-crystallized isomorphously with the native structure. The difference Fourier maps shows clear density for the ligands, unambiguously defining the active and allosteric sites. The complexes refinement produced the same conformation of the native, within experimental error. The allosteric sites are located at the interfaces of adjacent monomers from each trimer and the active sites (or catalytic) lie at the external side of each monomer, at the C-terminal end of the central parallel &#946-sheet. The monomer has a similar folding topology as a typical NAD binding domain, excluding the segments of aminoacids 135, 145-188 and 243-266. The native and complexes structures are at the allosteric state R concerted with MWC model for a K-system (Monod et al, 1965). A similar allosteric mechanism is found in the enzyme phosphofructokinase (Evans, 1981). A catalytic mechanism is proposed for the isomerisation-deamination reaction of the enzyme from general mechanism for aldo-keto isomerases.

A enzima Glucosamina-6-fosfato desaminase (GlcN6P desaminase) é envolvida na conversão reversível da D-glucosamina-6-fosfato (GlcN6P) em Fru6P e amônia, como parte do caminho metabólico de aminoaçúcares como fonte de energia celular. A enzima hexamérica (peso mol. 178200) exibe uma cooperatividade homotrópica intensa em direção à GlcN6P a qual é modulada alostericamente pelo ativador N-acetil-D-glucosamina 6-fosfato (GlcNAc6P). A GlcN6P desaminase foi cristalizada no grupo espacial R32, com parâmetros de rede a = b = 125.9 &#197 e c = 223.2 &#197 e um conjunto de dados à 2.1 &#197 de resolução foi coletado usando radiação de luz síncrotron (Horjales et ai., 1992). A procura no banco de dados de seqüências OWL não mostrou homologia significante com qualquer outra família de proteína, desta maneira a determinação da estrutura foi feita pela técnica de substituição isomórfica múltipla (MIR) a partir de dois derivados, um composto de platina, o K2PtCl4 e um complexo de mercúrio, o ácido mersálico. O mapa MIR a 3 &#197 de resolução mostrou contornos claros e utilizando técnicas de nivelamento de solvente (solvent flattening) estendeu-se as fases até 2.5 &#197. A enzima cristaliza-se com dois monômeros na unidade assimétrica. A densidade eletrônica final foi interpretada com o auxílio do programa gráfico \'O\', sendo possível determinar sem ambigüidade 230 dos 266 resíduos de cada monômero; a partir daí foram usados subseqüentes mapas de Fourier diferença para a localização de todos os outros resíduos. O refinamento do modelo foi feito utilizando o programa X-PLOR (Brünger, 1993), usando a rotina simulated annealing, obtendo o fator R final de 17.4% com 348 moléculas de água e quatro íons inorgânicos de fosfato. O enovelamento do monômero tem uma estrutura do tipo &#945/&#946 com uma folha-&#946 pregueada paralela central com sete fitas com topologia 4x, 1x, 1x, -3x, -1x, -1x, envolvida por ambos os lados por oito hélices-&#945 e uma hélice 310 com duas voltas. A sexta fita da folha-&#946 central tem um prolongamento no C-terminal que faz parte de uma segunda folha-&#946 antiparalela de três fitas com topologia 2, -1. O hexâmero tem uma simetria local 32, com dois trímeros empacotados frente-a-frente com uma rotação relativa de 15&#176 em tomo do eixo de ordem 3 e ligados por pontes salinas e algumas interações hidrofóbicas em tomo do eixo não cristalográfico de ordem 2. As moléculas de cada trímero formam um contato não usual de três resíduos Cis 219 próximo ao eixo de ordem três. Os complexos com ativador alostérico (GlcNAc6P) e inibidor competitivo (2-desoxi 2-amino glucitol 6-fosfato) foram co-cristalizados isomorficamente com a estrutura nativa. Os mapas Fourier diferença mostram claramente densidades para os ligantes, definindo sem ambigüidade o sítio ativo e alostérico. O refinamento dos complexos produziu a mesma conformação da proteína nativa, na margem de erro experimental. Os sítios alostéricos (seis) estão localizados na interface adjacente dos monômeros de cada trímero e os sítios ativos (ou catalíticos) no lado externo de cada monômero, no C-terminal da folha-&#946 central. O monômero tem uma topologia com enovelamento similar a um domínio de ligação de NAD, excluindo os segmentos de aminoácidos 1-35, 145-188 e 243-266. As estruturas dos complexos e da nativa estão em um estado alostérico R em concordância com o modelo MWC para um sistema do tipo K (Monod et al, 1965). Um mecanismo alostérico similar ao da GlcN6P desaminase é encontrado na enzima fosfofrutoquinase (Evans, 1981). Um mecanismo catalítico é proposto para a reação de isomerisação-desaminação da enzima GlcN6P desaminase a partir do mecanismo geral para aldose-cetona isomerases. / The enzyme Glucosamine-6-phosphate deaminase (GlcN6P deaminase) is involved in the reversible conversion of D-glucosamine-6-phosphate (GlcN6P) into Fru6P and ammonia. The hexameric enzyme (mol.wt.=178200) exhibits an intense homotropic co-operativity towards GlcN6P which is allosterically modulated by the activator N-acetyl-D-glucosamine 6-phosphate (GlcNAc6P). The GlcN6P deaminase was crystallized in space group R32, with cell parameters a=b= 125.9 &#197 and c = 223.2 &#197 and a native dataset was collected to 2.1 &#197 resolution at a synchrotron source (Horjales et al, 1992). A search of the OWL sequences database has shown no significant homology with any other known protein family. Therefore, the structure determination will have to be achieved through the Multiple Isomorphous Replacement technique from two isomorphous derivatives, a platinum compound K2PtCl4 and a mercury complex, mersalyl acid. The MIR map at 3 &#197 resolution showed clear molecular boundaries and solvent flattening techniques (Wang, 1985) were used to extend the phase set to 2.5 &#197. The final electron density map was interpreted with the aid of the graphic program \'O\'. The enzyme crystallizes with a dimmer in the asymmetric unit and 230 out of the total 266 residues of each crystallographically independent monomer could be unambiguously identified in the map. The remaining residues were located after subsequent difference Fourier maps. The refinement was made with program X-PLOR (Brunger, 1993), using the simulated annealing routine, obtained R=17.4 % with 348 water molecules and four inorganic phosphate ions. The monomer fold shows an &#945/&#946 structure with a central 7-stranded &#946-sheet with topology 4x, 1x, 1x, -3x, -1x, -1x, surrounded on both sides by eight &#945-helices and 2-turn 310 -helix. The sixth strand of the central &#946-sheet is common to a second 3-stranded anti-parallel &#946-sheet with topology 2, -1. The hexamer has local 32 symmetry, with two trimmers packed in a face-to-face arrangement with a relative rotation of 15&#176 around the 3-fold axis, and linked together by salt-bridge and some hydrophobic contacts. The molecules of each trimmer have extensive contacts and show an unusual feature of the three Cys219 residues closely clustered around the 3-fold axis. The complexes with allosteric activator (GlcNAc6P) and inhibitor (2-deoxy-2-amino glucitol 6-phosphate) were co-crystallized isomorphously with the native structure. The difference Fourier maps shows clear density for the ligands, unambiguously defining the active and allosteric sites. The complexes refinement produced the same conformation of the native, within experimental error. The allosteric sites are located at the interfaces of adjacent monomers from each trimer and the active sites (or catalytic) lie at the external side of each monomer, at the C-terminal end of the central parallel &#946-sheet. The monomer has a similar folding topology as a typical NAD binding domain, excluding the segments of aminoacids 135, 145-188 and 243-266. The native and complexes structures are at the allosteric state R concerted with MWC model for a K-system (Monod et al, 1965). A similar allosteric mechanism is found in the enzyme phosphofructokinase (Evans, 1981). A catalytic mechanism is proposed for the isomerisation-deamination reaction of the enzyme from general mechanism for aldo-keto isomerases.