ORIGINS AND DIVERSITY OF THE AGING REACTION IN PHOSPHONATE ADDUCTS OFSERINE HYDROLASE ENZYMES - WHAT CHARACTERISTICS OF THE ACTIVE-SITE DOTHEY PROBE

Molecular mechanics and dynamics combined with semiempirical calculati ons were carried out for purposes of comparison of the active site cha racteristics of AChE,(1) trypsin, and chymotrypsin as probed by their diastereomeric adducts with 2-(3,3-dimethylbutyl) methylphosphonofluor idate (soman), methylphosphonate monoester anions, and tetravalent car bonyl intermediates of the reactions of the natural substrates in each case. Glu199 is a key residue in the electrostatic catalytic mechanis m of AChE, in removal of the leaving group, and possibly by acting as an alternate general base catalyst. ''Pushing'' of an alkoxy ligand by Glu199 and the numerous small van der Waals interactions promote deal kylation in phosphonate adducts of AChE much more effectively than any other enzyme. A high concentration of negative charge created by the phosphonate ester monoanion and Glu199 adjacent to it fully accounts f or the resistance to the attack of even the strongest nucleophile appl ied for enzyme reactivation. Stabilization of the developing negative charge on the phosphonates in the soman-inhibited PSCS adducts of seri ne hydrolases is by electrophilic residues in the oxyanion hole (AChE) and the protonated catalytic His, P-R diastereomers of soman-inhibite d AChE can be accommodated in an orientation in which the oxyanion hol e interactions are lost and for which the stabilizing interactions are 17-26 kcal/mol smaller than in the P-S diastereomer.(2) The dealkylat ion reaction is almost equally likely in all diastereomers of soman-in hibited AChE, The stabilizing interaction energies are similar to 4 kc al/mol greater in the P-R than in the P-S adducts of the soman-inhibit ed serine proteases, There is 0.60 unit greater partial negative charg e on the phosphonyl fragment in the anion of phosphonate monoesters of Ser than at the oxygens of tetravalent carbonyl transients resulting in similar to 12-22 kcal/mol greater stabilization of the former than the latter.