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Marco Marin Tesis.Pdf UNIVERSITAT DE VALENCIA Departamento de Bioquímica y Biología Molecular INSTITUTO DE BIOMEDICINA DE VALENCIA CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS Máquinas moleculares que sintetizan anhidridos fosfóricos Memoria presentada por CLARA MARCO MARÍN para optar al grado de Doctora en Biología Director Prof. Vicente Rubio Zamora Valencia, Noviembre de 2006 i ii La autora ha disfrutado de becas FPI de la Generalitat Valenciana e I3P CSIC-Bruker España S.A. y de un contrato de Titulado Superior con cargo al proyecto BFU2004-05159. Es miembro del CIBER de Enfermedades Raras (CIBERER) del Instituto de Salud Carlos III. El trabajo se ha financiado principalmente con las ayudas BMC2001-2182 y BFU2004-05159 del Plan Nacional de Investigación Científica y Técnica (MCT y MEC). También se ha desarrollado bajo los auspicios del Instituto de Salud Carlos III (MSC; Redes C03/08, G03/54 y FISS PI052838). La autora agradece a los sincrotrones de Grenoble (ESRF) y Berlín (BESSY) por su apoyo en la recogida de datos y a la Unión Europea y al Ministerio de Ciencia y Tecnologia por la asistencia financiera para el uso de los mismos. iii iv AGRADECIMIENTOS Quiero dar la gracias a todas las personas que han contribuido a que este trabajo de tesis sea un objetivo alcanzado. A mi director de tesis, el Profesor Vicente Rubio, al que le debo no sólo la oportunidad de trabajar en su laboratorio sino también lo mucho que he aprendido a su lado. Gracias por hacerme partícipe de tus ideas y por estar dispuesto a discutir las mias. Gracias a todos mis compañeros del laboratorio, que han sido muchos: Andrea, Amparo, Belén, Benito, Chelo, Elena, Enea, Fernando, Jorge, Jose Luis, Juanma, Leonor, Lourdes, Matxalen, Mariano, Mari Luz, Michelle, Patricia, Rebeca, Sandra y Santi. Con cada uno de vosotros he compartido momentos diferentes, pero de todos he aprendido mucho: a trabajar en el laboratorio, a afrontar situaciones, a solucionar problemas, e incluso a conocerme mejor a mi misma. Tengo la suerte de haber hecho grandes amigos, y espero que nuestra amistad dure mucho tiempo. A todo el personal del Instituto de Biomedicina de Valencia. A los investigadores, becarios y personal del laboratorio, muchas gracias a todos por la convivencia en estos años, por vuestra ayuda siempre que la he necesitado. Al personal de mantenimiento, administración, informática, seguridad, limpieza …, por solucionar los pequeños desastres y por facilitarnos el dia a dia. Al Profesor Ignacio Fita, por su inestimable ayuda y apoyo, por hacer que la cristalografía sea más fácil. Al Dr. Alberto Marina, por su disponibilidad en todo momento. Al Dr. Javier Cervera y a su grupo, por permitirme colaborar con vosotros. A las Dras. Nuria Verdaguer y Cristina Vega por su dedicación y paciencia en la coordinación del BAG. A los miembros del laboratorio CRI2 del Instituto de Biologia Molecular de Barcelona por echarme un cable siempre que lo he necesitado y por su buena acogida durante mis visitas. A todos los investigadores que he conocido durante estos años, por participar en mi formación científica y en las inquietudes de los que estamos aprendiendo. A mis padres, mi hermana y mi marido, muchas gracias por vuestro interés, por vuestro apoyo incondicional, por compartir conmigo el entusiasmo y la euforia y por no dejarme flojear en momentos de desesperación. Gracias por motivame a estudiar y a trabajar en lo que realmente me apasiona y por apoyarme en las decisiones más importantes de mi vida. Muchas gracias. v vi ÍNDICE Introducción general ………………………………………………… 1 Objetivos ……………………………………………………………… 23 Capítulo 1 …………………………………………………………… 29 Site-directed mutagenesis of Escherichia coli acetylglutamate kinase and aspartokinase III probes the catalytic and substrate-binding mechanisms of these amino acid kinase family enzymes and allows three-dimensional modelling of aspartokinase. Capítulo 2 …………………………………………………………… 49 First-time crystallization and preliminary X-ray crystallographic analysis of a bacterial-archaeal type UMP kinase, a key enzyme in microbial pyrimidine biosynthesis. Capítulo 3 …………………………………………………………… 57 The crystal structure of Pyrococcus furiosus UMP kinase provides insight into catalysis and regulation in microbial pyrimidine nucleotide biosynthesis. Capítulo 4 …………………………………………………………… 77 A novel two-domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of Escherichia coli glutamate 5-kinase. Resumen de los resultados ………………………………………… 139 Discusión General ………………………………………………… 151 Conclusiones ……………………………………………………… 171 Bibliografía ……………………………………………………… 175 Correspondiente a la introducción general, resumen de resultados y discusión general. vii viii INTRODUCCIÓN GENERAL 1 2 Introducción general INTRODUCCIÓN GENERAL 1. OBJETO GENERAL DE ESTA TESIS Este trabajo de Tesis se ocupa de enzimas que sintetizan anhidridos del ácido fosfórico. Es imposible soslayar la crucial importancia biológica de estos compuestos, de entre los que la Figura 1 selecciona unos pocos. O O - H3C C O P O Acetilfosfato - O O O - H2N C O P O Carbamilfosfato - O OH O O O HO P O CH C O P OH 1,3-difosfoglicerato - - O O - O O O - C CH CH2 C O P O Aspartil-4-fosfato O + - NH3 O - O O O - C CH CH2 CH2 C O P O Glutamil-5- fosfato O + - NH3 O - O O O - C CH CH2 CH2 C O P O Acetilglutamil 5-fosfato O - NH O C O CH3 O O - Uridina Ribosa O P O P O - Nucleotidos di- y trifosfato O O - O P O - O Figura 1. Anhidridos del ácido fosfórico. El fosfato unido a cada una de las moléculas por un enlace anhidrido, se indica en rojo. Acetilfosfato y carbamilfosfato se encuentran entre los más simples y son posiblemente muy antiguos, objeto de síntesis prebiótica (Saygin, 1984) y precursores de otros compuestos en el largo periodo entre la sopa primigénia y el desarrollo de la vida, poseyendo el acetilfosfato un papel clave en el flujo global de carbono en la 3 Introducción general biosfera (Buss et al., 2001). El 1,3-difosfoglicerato, un compuesto intermedio de la glicólisis (Michal, 1999), tiene un lugar clásico entre los anhidridos mixtos carboxílico- fosfóricos. No resaltaré por conocido el papel de los nucleótidos di y trifosfato, de entre los que destaca particularmente el ATP por su papel de moneda energética universal en los seres vivos. También el NAD, el NADP y el coenzima A cumplen funciones indispensables en las oxi-reducciones y en la transferencia biológica de acetato (Michal, 1999) y contienen anhidridos fosfóricos. (B) (A) OH OH OH O O O O O O O Adenina HO P O P O P O NH R O NH2 2 NH2 OH ATP ADP OH OH OH + NH4 NH2 ATP Aminoácido OH OH Pi O O H2N PP OH O O i - O O P O O R Adenina - O O P O O NH OH Aminoacil-AMP 2 L-Glutamato L- g-Glutamil L-Glutamina fosfato tRNA OH OH (C) Carbamato ATP NH3 P OH ADP - OH i OH Adenina O OH - O O P O H N O Aminoacil-tRNA 2 - O NH O 2 O O O ATP Bicarbonato ADP O O R - O P OH O - O O P O Carbamilfosfato O H2N O ATP ADP (D) - OH OH O - O O P O - O O O Bicarbonato CARBOXILASA BIOTINA O C Pi SUSTRATO - O O CARBOXILASA BIOTINA C - O SUSTRATO Figura 2. Reacciones catalizadas por las aminoacil-tRNA sintetasas (A), la glutamina sintetasa (B), la carbamilfosfato sintetasa (C) y las carboxilasas dependientes de biotina (D) y que implican la formación de anhidridos fosfóricos como intermedios de la reacción. En rojo se representan los grupos que participan en la formación del anhidrido fosfórico. Las reacciones de síntesis biológica incluyen generalmente pasos en que se sintetizan anhidridos del ácido fosfórico, muchas veces como compuestos intermedios crípticos que son parte de los mecanismos reaccionales de sintetasas (ver ejemplos en la Figura 2), y otras veces como compuestos intermedios libres, producidos por un enzima 4 Introducción general y utilizados por el siguiente (Figura 1), aunque en ocasiones, como en el caso del g- glutamil fosfato, el papel del anhidrido es intermedio entre estas dos categorías, ya que aunque puede ser sintetizado por un enzima y utilizado por otro, es aún en ese caso posiblemente objeto de canalización entre el enzima productor y el aceptor (ver más adelante). En todo caso, en esta tesis me ocupo de cuatro enzimas que sintetizan estos anhidridos en forma libre, en un caso (N-acetil-L-glutamato quinasa) tangencialmente, en otro (aspartoquinasa III) realizando un estudio de mutagénesis-modelizado estructural y en los otros dos (UMP quinasa arqueal y glutamato 5-quinasa bacteriana) determinando la estructura tridimensional del enzima mediante cristalografía/difracción de rayos X. Los productos de tres de estos enzimas (acetilglutamato, aspartato y glutamato quinasas) son anhidridos carboxílico-fosfóricos, y por tanto estos tres enzimas pertenecen al grupo 2.7.2. de la Enzyme Commission, constituido por fosfotransferasas que usan un grupo carboxilato como aceptor (Figura 3). E.C. 2.7.2. Fosfotransferasas con un grupo carboxilo como aceptor 2.7.2.1 Acetato quinasa 2.7.2.2 Carbamato quinasa 2.7.2.3 Fosfoglicerato quinasa 2.7.2.4 Aspartato quinasa 2.7.2.6 Formato quinasa 2.7.2.7 Butirato quinasa 2.7.2.8 Acetilglutamato quinasa 2.7.2.11 Glutamato 5-quinasa 2.7.2.15 Propionato quinasa Figura 3. Enzimas del grupo 2.7.2 de la Enzyme Commission (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/2/). El listado ha sido modificado para excluir las entradas transferidas a otros grupos, la glutamato 1-quinasa, la acetato quinasa difosfato y la fosfoglicerato quinasa (GTP). En cursiva se señalan los enzimas de los que me he ocupado en este trabajo. El cuarto enzima (UMP quinasa) fabrica un anhidrido fosfórico-fosfórico (el del UDP) y por tanto pertenece al grupo de la E.C.
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