In the footsteps of an inhibitor unbinding from the active site of human carbonic anhydrase II

The crystal structure of human carbonic anhydrase (HCA) II bound to an inhibitor molecule, 6-hydroxy-2-thioxocoumarin (FC5), shows FC5 to be located in a hydrophobic pocket at the active site. The present work employs classical molecular dynamics (MD) simulation to follow the FC5 molecule for 1 mu s as it unbinds from its binding location, adopts the path of substrate/product diffusion (path 1) to leave the active site at around 75 ns. It is then found to undergo repeated binding and unbinding at different locations on the surface of the enzyme in water. Several transient excursions through different regions of the enzyme are also observed prior to its exit from the active site. These transient paths are combined with functionally relevant cavities/channels to enlist five additional pathways (path 2-6). Pathways 1-6 are subsequently explored using steered MD and umbrella sampling simulations. A free energy barrier of 0.969 kcal mol(-1) is encountered along path 1, while barriers in the range of 0.57-2.84 kcal mol(-1) are obtained along paths 2, 4 and 5. We also analyze in detail the interaction between FC5 and the enzyme along each path as the former leaves the active site of HCA II. Our results indicate path 1 to be the major exit pathway for FC5, although competing contributions may also come from the paths 2, 4 and 5. Communicated by Ramaswamy H. Sarma