Origins and Diversity of the Aging Reaction in Phosphonate Adducts of Serine Hydrolase Enzymes: What Characteristics of the Active Site do They Probe?

Abstract

Molecular mechanics and dynamics combined with semiempirical calculations were carried out for purposes of comparison of the active site characteristics of AChE, trypsin, and chymotrypsin as probed by their diastereomeric adducts with 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman), methylphosphonate monoester anions, and tetravalent carbonyl intermediates of the reactions of the natural substrates in each case. Glu199 is a key residue in the electrostatic catalytic mechanism 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 dealkylation 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 for the resistance to the attack of even the strongest nucleophile applied for enzyme reactivation. Stabilization of the developing negative charge on the phosphonates in the soman-inhibited PSCS adducts of serine hydrolases is by electrophilic residues in the oxyanion hole (AChE) and the protonated catalytic His. PR diastereomers of soman-inhibited AChE can be accommodated in an orientation in which the oxyanion hole interactions are lost and for which the stabilizing interactions are 17-26 kcal/mol smaller than in the PS diastereomer. The dealkylation reaction is almost equally likely in all diastereomers of soman-inhibited AChE. The stabilizing interaction energies are approximately 4 kcal/mol greater in the PR than in the PS adducts of the soman-inhibited serine proteases. There is 0.60 unit greater partial negative charge on the phosphonyl fragment in the anion of phosphonate monoesters of Ser than at the oxygens of tetravalent carbonyl transients resulting in approximately 12-22 kcal/mol greater stabilization of the former than the latter.

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“…95 The aged phosphylated cholinesterase cannot be reactivated due to several factors. 96,97 Firstly, the aged complex possesses a negative charge in the active site, making the attack of a negatively charged nucleophile more difficult. Besides, various non-covalent forces stabilize the aged complex: interactions among the OPC and the oxyanion hole, the acyl pocket and the histidine at the active site either stabilize the aged complex, either limit the dephosphylation by blocking the access of water or other nucleophiles to the phosphorus atom.…”

“…96 Finally, at least in the case of the nerve agent soman, it has been proposed the existence of a "push-pull" electrostatic mechanism, involving residues Glu-199, His-440, Trp-84 and the oxyanion hole in TcAChE, which would stabilize the carbenium formed during aging and favor a methyl migration that would transform a secondary carbenium in a tertiary one. 97,99,103,104 Spontaneous reactivation rates of phosphylated cholinesterases are determined by the structure of the phosphyl-enzyme. Except for the nerve agents, spontaneous reactivation occurs at clinically significant rates with the majority of the OPCs, but it is always slower than the deacetylation that happens in the hydrolysis of the natural substrate: Deacetylation of AChE occurs in microseconds, but dephosphylation in hours to days.…”

“…It is believed that the phenylalanine residues in the acyl pocket (mainly Phe-290 in TcAChE) are the major agents of this selectivity, due to the steric constraints imposed by them. 66,118 However, molecular modeling 97 and mutagenesis studies 63,119 have shown that the oxyanion hole, the anionic subsite and the peripheral anionic site also play an important role in stabilization of the phosphyl-AChE complex and in stereoselectivity. For nerve agents, the stereoisomer with the absolute configuration shown in Figure 9 is usually the most active AChE inhibitor, since the R'O group is greater than the R group.…”

“…113 This configuration corresponds to the S-isomers of sarin and VX, and to S-configuration around the phosphorus atom (P S ) for soman. 63,97,118 Table 3 shows the effect of the stereochemistry of nerve agents on their anticholinesterase activity and acute lethality, which is not very expressive. 97 In the particular case of soman, there is low selectivity related to the chiral carbon atom.…”

“…63,97,118 Table 3 shows the effect of the stereochemistry of nerve agents on their anticholinesterase activity and acute lethality, which is not very expressive. 97 In the particular case of soman, there is low selectivity related to the chiral carbon atom. In fact, the P S C S isomer is a bit more toxic than the P S C R , and there is practically no difference between their aging rates, 120 probably because the same carbenium is formed in both cases.…”