| Abstract: | Molecular dynamics simulations of the serine protease porcine pancreatic elastase in the native state (in vacuo and solvated) and complexed to two trifluoroacetyl dipeptide inhibitors are reported. Crystallographically resolved water molecules were included in all calculations, and for two of the simulations the active site was solvated through application of a "dome" of water molecules. The form of the interaction potential energy function and a procedure for deriving parameters to use with it are discussed. Comparisons are made between the molecular dynamics results and available high-resolution X-ray data with respect to radii of gyration, atomic positions, positional fluctuations, dihedral angles (φ, ψ and ω) and hydrogen bonding pattern. The magnitudes and time development of residue motions with respect to their involvement in particular secondary structure (α-helices, β-sheets, β-turns, loops) are also examined. Structural features of the simulations agreed well with the X-ray structure with the exception of residues involved in β-turn geometries. Some damping of motions was observed in the case of the solvated simulations. Positional fluctuations of residues in the free and bound states are compared and it is found that the bound molecules have a global increase in motion and that this increase is particularly dramatic in the S₂-->S₄ regions of the active site where the ligand molecule is bound. In hopes of rationalizing the high inhibitory power of these nonproductively bound ligands, the active site dynamics (involving the serine-histidine-aspartate triad) of the solvated native enzyme and the trifluoroacetyl-l-lysyl-l-alanyl-p-trifluoroisopropyl anilide complex were examined. Crystallographic and NMR results indicate a conformational shift of the catalytic histidine residue such that it exhibits an improved hydrogen bonding interaction with the catalytic serine residue in the active site. Because this shift does not occur with the less potent acetylated inhibitors, this was thought to be related to the mode of inhibition of the trifluoroacetyl inhibitors. Therefore, time series of the geometrical properties of the hydrogen bond were used to track the strength and pattern of formation of the interaction between serine and histidine. Results indicate that spectroscopically and crystallographically observed shifts are real and are consistent with a catalytic mechanism in which a proton is shuttled from serine to histidine to the leaving group, rather than the direct transfer of the hydrogen from serine to the leaving group. |
|---|