The metalloenzyme Escherichia coli (E. coli) Alkaline Phosphatase (AP) has a homo-dimeric quaternary structure, which is essential for the enzyme to achieve its catalytic activity, the acceleration of phos- phoester hydrolysis. Employing computational approaches, at differ- ent levels of complexity and sophistication, this thesis aims at under- standing the interplay of structure, dynamics and function of this im- portant enzyme, that is responsible for the supply of vital inorganic phosphate. At low resolution, coarse grained models of the enzyme are con- structed, using an Elastic Network Model (ENM), to explain the in- fluence of the global dynamics, defined by the quaternary structure of the enzyme, on its functionality. Comparative analysis of the col- lective motions, of individual subunits within a homo-dimer of apo and holo enzymes, allows us to interpret the experimental proposal of negative cooperativity. The intrinsic asymmetry of the subunits within the dimer, is already encoded in the enzymes three-dimensional structure, as becomes apparent from the normal mode analysis of the low resolution ENMs, with alternate opening and closing motions present in only one subunit at a time. These results, in conjunction with the analysis of Molecular Dynam- ics (MD) simulations of the atomistic models, at the nanosecond time scale, suggest a mechanical coupling between the correlated motions of individual subunits and their active sites, via the dimer interface. The negative cooperativity of the subunits is further explained by the analysis of multiple, independent MD simulations that reveals subtle differences in the hydrogen bonding network of the subunits and dy- namics of their active site residues, demonstrating a distinct asymmet- ric behaviour, with increased flexibility of one subunit versus rigidity of a second one. Information about structural changes is transmitted between the subunits via the hydrogen bonding network across the in- terface. At least two such communication pathways can be proposed, based on the analysis of MD simulations, both via the interfacial alpha- helix containing residues T559 and T555 that are hydrogen bonded to the residue N416, which is in turn, connected to the residues in the active site. Analysis of the MD simulations confirms that, while the active site of one subunit retains the inorganic phosphate product, as demon- strated by the small changes in the distances between the active site residues, another subunit is "letting the product go" in order to make the active site available for another turnover of substrate binding. ￼vii Analyses of the MD simulations further suggest that correlated mo- tions of the monomeric subunits of the dimer, as well as the dynamics, and hence the architecture of the active sites, play an important role in the functionality of Alkaline Phosphatase. Although each subunit of the enzyme is equipped with its own catalytic sites, a monomeric AP does not exist in nature and the engineered mutants have significantly reduced activity. Due to the absence of a crystal structure, a model of a monomeric form of the enzyme is constructed based on the crys- tal structure of the apo dimer. In addition, a T59R mutant is built, where interface residues T59 and T559 are substituted by a bulky and charged Argenine residues. Experimental evidence suggests that such a substitution destabilizes the dimeric interface, resulting in a separation into isolated monomers, with reduced structural stability and catalytic activity. Our MD simulation results confirm that the over- all dynamic behaviour of the monomer is different from that of the corresponding dimer and resembles more that of the T59R mutant. Furthermore, comparative analysis based on MD simulations of di- meric and monomeric forms of AP reveals important structural and dynamic features enabling the native dimer to be catalytically func- tional. The stabilisation provided by the interface of the two subunits in the dimeric form of AP is found to be essential for a catalytically competent structure of the active sites. Breaking of the hydrogen bond between residues Y402 and D330, that are located near the active site, as observed in the MD simulations of the monomer, results in the incorrect positioning of the catalytically important, divalent, zinc ion. Understanding the nature of the correlated motions of the subunits within the dimer, and their connection to the enzyme’s activity is an important step in completing our knowledge on structure-dynamics- function relationship of E. coli Alkaline Phosphatase and related en- zymes. Our findings confirm that the structural stability of dimeric AP, provided by the hydrogen bonding network across the interface, is essential for the enzymatic activity. By a combination of different computational approaches, we gained an in-depth understanding of the relationship between the enzymes’ dimeric quaternary structure and its functionality.
|Supervisor:||Smith, Prof. Jeremy C.|
|Place of Publication:||Heidelberg, Germany|
|Date of thesis defense:||5 March 2013|
|Date Deposited:||02 Apr 2013 12:41|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences|
|Subjects:||570 Life sciences|
|Controlled Keywords:||Quaternary structure, Enzymatic activity, Molecular dynamics simulation|