Dissociation constants of five oximes with native and OP conjugated hAChE are summarized in the . Constants for interaction with native hAChE determined in both, substrate competition experiments measuring enzyme activity and stopped-flow experiments measuring direct binding, agreed well and were therefore averaged and presented as one constant in the table. The binding of three triazole oximes to native AChE with dissociation constants in low micromolar range was significantly, one to two orders of magnitude, greater than that of 2PAM and HI6 reflecting a better fit of elongated and slightly bent triazole structures () within the long, narrow and slightly curved active center gorge of AChE. Conjugation of OP moieties with the active serine upon AChE inhibition by VX, paraoxon (POX) and cyclosarin (CS) resulted in an increase in Kd values for all oximes reflecting a general reduction in the available binding space or distortion [9
] in the active center gorge of phosphylated hAChE. The extent of Kd
increase was consistent with the increase of molecular volume of covalently attached moieties (calculated using DS Visualizer by Accelrys as a volume of solvent accessible surface generated with a sphere of 1.4 Å radius), in the following order : VX-cojugate (~ 79 Å3
), POX-conjugate (~ 95 Å3
) and CS-conjugate (~ 115 Å3
). Dealkylation of VX or CS inhibited enzyme yields an aged form of hAChE where ~ 54 Å3
of the gorge volume is taken by an anionic OP moiety. Binding of all five oximes to aged hAChE was much tighter than to any other OP-hAChE conjugate (). In fact Kd
s for oximes 28B, 2PAM and HI6 were one order of magnitude lower than corresponding Kd
s for binding to non-conjugated, native hAChE. This decrease in Kd
s in spite of ~ 54 Å3
reduction of the gorge volume available for oxime binding points to significant stabilizing role of electrostatic interaction between electron deficient pyridinium oxime moieties and negatively charged methylphosphono anion of the aged hAChE. Only for oximes 81A and 153 that bound to native hAChE tightest of all five oximes, binding to aged hAChE was not further improved. The aged OP-hAChE conjugate thus provides tightest stabilization for oxime binding, narrowing down 375-fold range of Kd
values (0.4 uM – 150 uM) observed for binding to the native hAChE ten–fold, to a 38-fold range (0.23 uM – 8.7 uM for the aged hAChE). Recent structural studies are consistent with this observation showing that both 2PAM and HI6 bind to aged AChE in orientations different from those found bound to native AChE (). HI6 for example binds only to the upper half of the native mouse AChE gorge, above the “choke point” (), while it was found bound to the very base of the aged mouse AChE gorge (). Also, 2PAM orientation bound to native Torpedo californica
AChE () was different from the one found bound to the aged Torpedo
AChE (). The inability of 2PAM and HI6 to reactivate aged AChE was thus not due to the lack of oxime binding in the proximity of the aged OP moiety in the AChE active center gorge. In addition to the inherent stability of the dealkylated OP conjugates, the presumed unproductive oxime group orientations directed away from phosphorus atom in both aged enzyme * oxime complexes () account for the lack of reactivation.
Table 1 Dissociation constants (Kd) for binding of oximes to native and OP-conjugated hAChE. Constants were determined by one or more different experimental approaches described in Material and Methods section, and averaged with standard deviation lower than (more ...)
Structures of oxime AChE reactivators used in this study.
Figure 2 Reversible complexes of 2PAM and HI6 with native and aged AChE. Crystal structures of HI6 bound to the active center gorge of A) native mouse AChE (pdb ID 2GYU, ) and B) aged mouse AChE at S203 (pdb ID 2WHQ, ). Crystal structures of 2PAM bound (more ...)
Kinetics of oxime binding to native and OP conjugated hAChE was also studied, but only for hAChE*oxime complexes with Kd
values lower than 8 μM, since the approach to equilibrium for other, weaker complexes could not be resolved in the stopped-flow apparatus. The second order association rate constants were therefore determined for binding of three triazole oximes with native, VX inhibited and aged hAChE. The constants varied between 1.2 and 7.7 × 109
, characteristic for protonated ligands such as tacrine and 9-aminoacridine, and slower than quaternary ligands carrying a permanent positive charge [6
]. Since oximes 28B and 153 are bisquaternary and 81A monoquaternary, permanently charged ligands, their slower association rates could be attributed to the influence of deprotonated oxime moieties. These oximes thus may associate with hAChE with their oxime groups deprotonated and positioned for nucleophilic attack on the conjugated phosphorus atom. On the other hand static, structural data obtained typically in crystallization media of pH lower than 7.0 point to possibility of hydrogen bond formation between oxime and catalytic triad His [12
] suggesting that at pH ≤ 7 oximes bind to OP conjugated AChE in their protonated state. The association rate constants of oximes 28B and 81A show modestly diminished rates for aged and VX-inhibited hAChE. The influence of decrease in the available binding space in the hAChE active center gorge on ligand association kinetics was also manifested in the association rate constants of 9-aminoacridine ().
Table 3 Interaction kinetics of 9-aminoacridine with native and OP-conjugated hAChE. The second order association constants (k1) were determined as described in Material and Methods section. The calculated standard deviation was lower than of 30% of the constant (more ...)
In summary results of our study indicate that conjugation of hAChE with large OPs results in altered geometry of the active center gorge and compromised binding of oxime reactivators leading to loss of their reactivating potency. Dealkylation of OP-conjugated hAChE resulting in aged hAChE enhanced oxime binding, but according to structural studies, in completely nonproductive orientation for reactivation. The electrostatic interactions between anionic aged hAChE and cationic oximes thus resulted in further stabilization rather than in destabilization and breakdown of otherwise nonreactivatable aged hAChE.