PROBING THE ACTIVE-SITE OF ACETYLCHOLINESTERASE BY MOLECULAR-DYNAMICSOF ITS PHOSPHONATE ESTER ADDUCTS

Molecular dynamics (MD) simulations using CHARMM were performed for th e solution structures of the pentacoordinate and tetracoordinate PSCS and PRCS adducts of Torpedo californica (Tc) acetylcholinesterase (ACh E) formed with 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) to assess the molecular origins of stereoselectivity of phosphonylati on. MD simulations were also carried out for the PSCS transients in so man-inhibited trypsin to evaluate the differences in the mode of opera tion of the two enzymes. Parameters for the pentacoordinate phosphonat e fragments were constructed from results of an nb initio calculation at the 6-31G level for a model compound, and those for the tetracoord inate phosphonate fragments were from MNDO calculations. Starting equi librium structures for the above and for analogous structures for chym otrypsin were generated and energy-optimized in program YETI. The ster eoselectivity of AChE for the levorotatory diastereomers of soman amou nts to >5.6 kcal/mol difference in transition state free energies and can be rationalized based on the results of the MD calculations: There is a predominant conformation of transient forms of the PsCs diastere omer of soman:inhibited AChE in which every ligand in phosphorus is st abilized by an optimal binding feature of the active site. In contrast , the phosphonyl fragment in the PRCS diastereomer may be accommodated with equal difficulty, at least, two different ways: In the most favo rable conformation, the phosphoryl oxygen is engaged in weak interacti ons with constituents of the oxyanion hole if adjustments in the Ca ba ckbone and substantial motions of Trp84, Trp233, Phe288, and Phe290 ar e allowed. The remarkable efficiency of F- departure from the pentacoo rdinate transition states of phosphonylated AChE cannot be explained b y general base catalysis by HisH(+)440. Leaving group departure from t hese structures must be promoted by electrostatic forces, ''push'' fro m Glu199 and ''pull'' from the oxyanion hole, in addition to steric st rain. One of the distinguishing features of the crystal structure of T cAChE is the short H-bonds in the catalytic triad. The His440 N delta- --OOC beta Asp327 bond distance is 2.5 W (2.8 Angstrom resolution) in AChE and 0.2 Angstrom shorter than the corresponding H-bond in trypsin and chymotrypsin (1.5 Angstrom resolution). This distance increased t o 2.7 Angstrom during the dynamics simulation. However, the average H- bond distances are further shortened by 0.05-0.3 Angstrom in energy-mi nimized structures of the adducts of AChE covalently modified by soman at the pentacoordinate and tetracoordinate intermediate stage. MD sim ulations of the optimized structures of native AChE and its adducts ga ve insight into how the skeletal motions accommodate an overcrowded ac tive site particularly in the pentacoordinate adducts. The steric reli ef is only partial and is balanced by a repositioning of Glu199 toward the catalytic triad and phosphonyl fragment. This subtle reorientatio n of active-site residues should be relevant to the prominent catalyti c efficiency of AChE.