Please use this identifier to cite or link to this item: http://hdl.handle.net/2445/42881
Title: Molecular Modeling of Enzymes. Application to the Study of Phosphoryl Transfer Reactions and the Dynamics-Function Relationship.
Author: Marcos Benteo, Enrique
Director: Crehuet, Ramon
Solé, Albert (Solé Sabaté)
Keywords: Quantum mechanics
Molecular dynamics
Brownian dynamics
Phosphorane
Diffusion
Crowding
Thermostability
Electric fields
Mecánica cuántica
Mecànica quàntica
Dinàmica molecular
Dinámica molecular
Dinàmica browniana
Issue Date: 16-Jan-2012
Publisher: Universitat de Barcelona
Abstract: [eng] Enzymes are the most proficient catalysts that evolution has developed to assist the chemical reactions that make life possible. By means of molecular simulations the present thesis addresses three aspects of fundamental importance for the enzymatic function: reactivity, dynamics and thermostability. The reactivity studies focuses on phosphoryl transfer reactions, which are involved in a broad range of biological processes. We have studied intermediate pentacoordinated species (phosphoranes) being formed in the course of a nucleophilic substitution at phosphorus with quantum mechanical methods (QM). The striking feature of these compounds is that they can be strongly polarized due to the dative character of the apical bonds. Thus external electric fields can alter the geometry and stability of these compounds to the extent that the reaction mechanism can be modified. Indeed, enzyme active sites exhibit strong electric fields able to introduce such effects. The knowledge acquired in model systems of pentacoordinated phosphorus has been applied to evaluate with high-level quantum mechanical/molecular mechanics (QM/MM) methods the reaction path of a phosphoryl transfer in the controversial beta-phosphoglucomutase enzyme. Our calculations show that a pentacoordinated intermediate is not stable in this enzyme. This indicates that the X-ray structure of a complex of the enzyme with a potential phosphorane was wrongly characterized. Enzyme dynamics have been studied in the context of the amino acid kinase family of enzymes. We have analysed the large-amplitude motions associated to ligand binding process involved in catalysis and allosteric regulation of the activity. By means of elastic network models, we show that the shared fold of this family involves shared dynamical features associated to ligand binding events. We have also analysed how oligomerization modulates large-amplitude motions and determines the binding mechanism. Another important aspect of enzymes is their adaptation to specific temperatures. We have analysed the relationship between thermostability and dynamics for a thermo-mesophilic pair of enzymes that was studied by means of neutron scattering. In this experiment, the flexibility of a thermophilic enzyme was found to be less sensitive to temperature changes than its mesophilic homologue pointing to a novel mechanism of protein thermostability. To understand the origin of these results, we performed molecular dynamics simulations to describe intramolecular motions and Brownian dynamics (with a box of 1000 protein molecules) to account for crowding effects in solution. Our results show that the different thermal behavior of the two proteins arises from the different diffusional properties of the two enzymes, despite being similar in size and shape. This is due to the fact that the thermophilic enzyme exhibits a more intense electrostatic potential thus introducing differences in the inter-protein electrostatic interactions in the crowded solution that, of course, affect diffusion. This provides a new interpretation of the results obtained in the original experiment.
[spa] La presente tesis se centra en la modelización molecular de tres aspectos necesarios para la función enzimática: reactividad, dinámica y termostabilidad. Los estudios de reactividad se han centrado en reacciones de transferencia de fosfato, las cuales están implicadas en un amplio rango de procesos biológicos. Se han estudiado los potentiales intermedios pentacoordinados (fosforanos) que pueden formarse en sistemas modelo y se observa que son especies muy polarizables. Los campos eléctricos externos pueden alterar su geometría y estabilidad de forma que pueden llegar a alterar el mecanismo de reacción. Con este conocimiento previo se ha estudiado con métodos QM/MM el controvertido intermedio pentacoordinado del enzima beta-fosfoglucomutasa. Los cálculos confirman que dicha especie no es un fosforano estable y que, por lo tanto, la estructura cristalográfica no fue resuelta correctamente en concordancia con recientes pruebas experimentales. El estudio de la dinámica se ha focalizado principalmente en los movimientos lentos de gran amplitud que están asociados a procesos de unión de sustrato y regulación de la actividad por alosterismo. Esta parte se ha centrado en la familia de las quinasas de aminoácido que presentan importantes cambios conformacionales asociados a su función biológica y que han sido muy bien caracterizadas por cristalografía. Mediante modelos de red elástica, se ha confirmado que la similitud estructural de los miembros de esta familia conlleva también similitud en la dinàmica de gran amplitud, la cual depende principalmente del plegamiento proteico. Además se han estudiado los efectos de oligomerización en la dinàmica de los miembros de esta familia. En tercer lugar, se ha analizado la relación entre termostabilidad y dinámica en el contexto de un experimento de dispersión de neutrones. Las simulaciones de dinámica molecular, para estudiar la flexibilidad interna, y de dinámica Browniana, en una caja de 1000 proteinas en disolución para tener en cuenta la difusión en “crowding”, muestran que el diferente comportamiento térmico de la movilidad que se observa en el experimento no se debe a la flexibilidad interna, sino a la distinta difusión que presentan el enzima termófilo y su homólogo mesófilo por sus diferentes interacciones electrostáticas en la superficie.
URI: http://hdl.handle.net/2445/42881
Appears in Collections:Tesis Doctorals - Departament - Química Física

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