| Popis: |
Mechanistic details of events preceeding and occurring during enzymatic catalysis are extremely difficult to obtain experimentally. While molecular dynamics methods permit the simulation of discrete steps along the catalytic pathway, they also can overwhelm the investigator with a mountain of structural details, necessitating the development of specific analytic tools. Based on recent crystallographic data, several molecular dynamics simulations have been performed using the AMBER 3.0 program package. Two different simulations for the hydrated native enzyme (100 and 50 ps) and two simulations (30 and 70 ps) of the Michaelis complex of this enzyme with the substrate (Thr-Pro-nVal-Leu-Tyr-Thr) have been performed. Dynamical properties of the active site (especially the catalytic tetrad: Ser-195, His-57, Asp-102 and Ser-214; chymotrypsinogen numbering system) have been examined using the program MD_ANALYSIS_1. It, together with the program MDKINO, facilitates analysis of dynamical changes of conformation (especially the hydrogen bond network) of the active site. Hydrogen bonding among Asp-102, Ser-214 and His-57 was quite stable, but the catalytic Ser-195 sidechain was flexible. Therefore the catalytically crucial H-bond between HO γ (Ser-195) and N ϵ (His-57) is relatively labile. In the native case (i.e. without substrate) this H-bond was never formed due to competition for the acceptor atom (N ϵ ) between water molecules and the HO γ group. In the Michaelis complex the H-bond is more readily formed, although the sidechain (Ser-195) may sometimes change its conformation do to the influence of the carbonyl group of Ser-214. Due to the dynamical motion of the enzyme there were six different short periods in which the distance between both heavy atoms in this crucial H-bond was less than 2.6 A, which may facilitate proton transfer from Ser-195 to His-57 (the first step during the proper catalysis). These results suggest mechanistic details about the precise clockwork of a functioning enzyme. |
