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FU Berlin | |
Peter Vagedes :Simulation enzymatischer Reaktionen |
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AbstractAbstractIn this work the role of electrostatic interactions in proteins for different processes were investigated. The main subject was to study the influence of electrostatic interactions on catalysis and protein-protein association.The protonation pattern of the enzyme acetylcholinesterase was determined by a Monte-Carlo titration method on the basis of the electrostatic potentials at the titratable groups. The calculated protonation pattern showed that not all titratable groups are in their standard protonation state. This finding suggested that the function of acetylcholinesterase has to be judged not only on its structure and the standard charge distribution, where generally titratable groups are assumed to be charged, but also on the carefully established protonation pattern. In acetylcholinesterase, especially the residue Glu199, which is near to the active site and highly conserved among different species, gave rise to several discussions, because the Gln199Ala mutation did not show as large effects on the reaction kinetics as one could expect when a charged group is replaced by alanine. My titration calculations showed that in fact Glu199 is not charged but protonated in the free as well as in the acylated enzyme. This explains very well the small effect in the mutation experiment. The next question was, if the uncharged protonation state of Glu199 is consistent with the catalytic mechanism of acetylcholinesterase. I investigated the rate determining deacylation step of acetylcholinesterase. This simulation study was done within the framework of the empirical valence bond method (EVB). With this method the free energy along the reaction path can be calculated. The unique parameterization facilities of the EVB method allow a meaningful comparison of the calculated free energies with experimental values, which are deducible from the measured reaction rates. In my study, I could reproduce the experimental reaction rate of the deacylation with a sufficiently small deviation: The rate obtained by the simulation studies was only by a factor of 30 smaller than the experimental rate. This result could only be obtained with acetylcholinesterase in the appropriate protonation state. As a confirmation of the previous Monte-Carlo titration study I found, that Glu199 in the charged titration state decreased the rate by the factor 10^4. This finding underlines the importance to consider the correct protonation pattern for theoretical investigations on enzyme functions. Moreover, the study on acetylcholinesterase revealed that the protonation pattern in the active site is in agreement with the general assumed mechanism of serine hydrolases, where the histidine of the catalytic triad forms a hydrogen bond with the Asp or Glu residue of the catalytic triad, that is negatively charged. In my study the proton at His440 was found on the right nitrogen atom to form this hydrogen bond and Glu327 was found to be negatively charged. The trajectory of the simulated reaction showed, that the tetrahedral intermediate of the deacylation step is stabilized by an oxyanion hole as is also known for the acylation step. I found in my calculations, that the presence or absence of choline, the reaction product of the initial acylation reaction, effects neither the protonation pattern of the enzyme nor the energetics of the catalyzed reaction. Hence, choline might still be in the binding pocket during deacylation. This is somehow surprising, as choline is positively charged and should therefore have an influence on the active site properties. This indifferent finding suggests, that choline may leave the binding pocket also after the deacylation step in contrast of what is generally assumed. Another field of application of electrostatic interactions in proteins is the protein-protein association process. An interesting system is the enzyme arylsulfatase A, that builds octamers at pH values around 5 and dissociates to dimers at pH values above 6. From the crystal structure, it was suggested that this pH dependent behavior is controlled by the protonation deprotonation equilibrium of Glu424, the only titratable group in the dimer-dimer interface. I investigated the titration behavior of arylsulfatase A, when the dimers are associated or isolated and found that indeed the protonation behavior of Glu424 differs significantly. The protonation probability is larger, when the dimers are associated. This was also suggested from the interpretation of the structure: As both Glu424 are only separated by around 5 Å in the dimer-dimer interface, it would be energetically unfavorable to have both in the charged state. The titration behavior of Glu424 also supports a second conclusion drawn from the x-ray structure determination experiments. Glu424 was found to have possibly two conformations: One conformation suitable for an intermolecular hydrogen bond that supports the dimer-dimer association, and one conformation suitable for an intramolecular hydrogen bond, preferably formed in the isolated dimers. The intermolecular hydrogen bond is formed with Glu424 protonated. The intramolecular hydrogen bond is formed with Glu424 unprotonated. The titration calculations showed, that the protonation probability of Glu424 in the dimer-dimer interface is significantly higher, when it is in the conformation suitable for the intermolecular hydrogen bond. To account for the pH dependent behavior of the dimer-dimer association of arylsulfatase A I calculated the free energy of association in dependence of the pH-value with two different methods: A Monte-Carlo method yielding the population of the associated and isolated state and the proton-linkage model. The first method relies on good reference energies of the associated and dissociated forms of the ASA dimer in solution. The computation of suitable reference energies is problematic. The calculations are therefore more qualitative and show, that the associated form is more stable at pH 5 than at pH 7. The main contribution to the association comes from hydrophobic interactions, which are only qualitatively considered in the calculations via a surface factor. Moreover it could be shown, that electrostatic interactions do not favor association but rather work against it. The proton linkage model provides the same pH dependence of the association energies. The last part of this work is on the mechanism of sulfate ester hydrolysis accomplished by arylsulfatase A. The mechanism may proceed via a gem-diol in the active site of arylsulfatase A. This unusual component in an enzyme is formed by hydration of an aldehyde. The hydration of the aldehyde has to be at least as fast as the overall rate of arylsulfatase A. This seems to be only possible if it is base- or acid-catalyzed. From titration calculations I suggest, that the hydration of the aldehyde is catalyzed by a lysine residue in the active site. This lysine residue was found to be unprotonated and could therefore act as a base. The systems investigated in this work show clearly that besides the structure of an enzyme, which is prerequisite to draw conclusions on its function, the protonation pattern has to be investigated, because it may have a large influence on the catalytic mechanism as well as on protein-protein association processes. Therefore, the electrostatic energies depending on the different protonation states must always be included, when quantitative structure activity relationships are investigated.
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Table of ContentsDownload the whole PhDthesis as a zip-tar file or as zip-File For download in PDF format click the chapter title |
More Information: | ||
| Online available: | http://www.diss.fu-berlin.de/2001/49/indexe.html | |
| Language of PhDThesis: | english | |
| Keywords: | enzymatic reactions, computer simulations, electrostatics | |
| DNB-Sachgruppe: | 30 Chemie | |
| Date of disputation: | 26-Mar-2001 | |
| PhDThesis from: | Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin | |
| First Referee: | Prof. Dr. Ernst Walter Knapp | |
| Second Referee: | Prof. Dr. Wolfram Saenger | |
| Contact (Author): | vagedes@chemie.fu-berlin.de | |
| Contact (Advisor): | knapp@chemie.fu-berlin.de | |
| Date created: | 05-Apr-2001 | |
| Date available: | 05-Apr-2001 | |
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