This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Adenylate kinase (AK) catalyzes the transfer of one phosphate group from ATP to AMP, producing two ADP as end products. Previously, the Kern's group determined that the conformational transition between the open and the closed states, rather than the chemical catalysis, is the rate limiting step of the reaction. Our main goal of this project is to determine the transition state structure(s) between the two states. The enzyme is made up of three domains: the core, the ATP and the AMP lid. In our previous work we have simulated Aquifex aeolicus adenylate kinase (AAK) in its ligand free form and in its inhibitor bound form. In both simulations, the motion of the ATP lid is independent of the motion of the AMP lid. Therefore a two-dimensional (2D) potential of mean force (PMF) computation is necessary to map the free energy landscape of the enzyme, rather than the one dimensional PMF computations carried out by other groups on AK homologues. Based on our previous simulation work, we have defined two geometrical angles between the centers of mass of selected groups of residues to be the two reaction coordinates. We have used the umbrella sampling algorithm to sample the conformations at some of the grid points between the two states. We need more CPU power to finish the simulations on the remaining grid points, and also to extend the trajectories on the grid points that are already sampled. Once all these simulations are completed, we will use weighted histogram analysis method (WHAM) to construct the 2D PMF surface. From the 2D PMF surface, we will first compute the free energy difference between the open and the closed states. Such a value has been experimentally determined by Kern's group. A direct comparison between the computed and the experimental values will be used to justify the computation protocol. We will also pinpoint the transition state structure(s), and the key interactions present in the transition state structure(s) and not in either the open or the closed state. We will then predict the mutations of which residues influence the transition rate between the two states. These predictions can then be further testified by experimental mutagenesis studies.
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