Molecular Modeling of Enzymes. Application to the Study of Phosphoryl Transfer Reactions and the Dynamics-Function Relationship.

• Autores: Enrique Marcos Benteo
• Directores de la Tesis: Josep Maria Solé i Sabaté (dir. tes.), Ramon Crehuet Simon (dir. tes.)
• Lectura: En la Universitat de Barcelona ( España ) en 2012
• Idioma: inglés
• Tribunal Calificador de la Tesis: Modesto Orozco López (presid.), Pau Bernado Pereto (secret.), Vicente Moliner Ibáñez (voc.)
• Materias:
  • Física
    • Química física
  • Química
    • Bioquímica
      • Proteínas
    • Química orgánica
      • Mecanismos de reacción
  • Ciencias de la vida
    • Biofísica
• Enlaces
  • Tesis en acceso abierto en:  TDX  Dipòsit Digital de la UB 
• Resumen

  • 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.