Inhibition of human D134H with paraoxon and an analogue of cyclosarin was 2-8 times slower than that of human wt (), whereas inhibition by soman and analogues of VX was not affected by D134H substitution. Introduction of additional substitutions in two double mutants further slowed inhibition up to two orders of magnitude.
Relative inhibition rates of hD134H wt; hD134H_E202Q and hD134H_F338A.
2PAM reactivation of paraoxon inhibited human D134H is, on the other hand, 6 times faster than human wt (). Our previous study indicated that differential occupation of the acyl pocket in mouse AChE, by covalent ligands of varying geometry, had distinct effects on spectra of fluorophores covalently attached on the AChE surface in the general spatial vicinity of residue D134 [8
]. In order to determine whether the acyl pocket stabilized ethoxy substituent on phosphorus in diethylphosphorylated hAChE was primarily responsible for the observed enhancement of reactivation rates, the enzymes were inhibited with an excess of racemic VX analogue. Inhibition was assumed to yield wt and D134H AChEs conjugated primarily by the SP
VX enantiomer with methylphosphonyl substituent of the conjugate stabilized in the acyl pocket and the ethoxy substituent oriented towards the choline binding site. Reactivation of both wt and D134H hAChE SP
VX cojugates with 2 PAM showed similar reactivation rates. This suggests that the 2PAM reactivation rate enhancement observed in the diethylphosphorylated (paraoxon inhibited) D134H mutant may be linked with improved stabilization of the ethoxy substituent in the mutant acyl pocket and is consistent with the observed three-fold enhancement of the HI6 reactivation rate (). To verify this hypothesis further 2PAM reactivation of the RP
IPMP inhibited enzyme conjugates (identical to RP
Sarin conjugated AChE) was studied but yielded no effect of D134H mutation. Thus, our experiments implicate differential flexibility of the acyl pocket in D134H substituted hAChE, but the actual mechanism of the 2PAM reactivation enhancement remains unclear.
Relative reactivation rates of OP inhibited hD134H; hD134H_E202Q and hD134H_F338A.
Human D134H_E202Q and D134H_F338A non-aging double mutants did not reactivate any faster than the wt or the hD134H mutant (). In fact, of all tested enzymes the one most compromised with mutations was D134H_E202Q mutant. It showed both inhibition and reactivation rates about 20 times slower in comparison to wt and D134H ( and ). The characteristics of double mutants mostly arose from E202Q mutation that has slow phosphonylation and reactivation rates. We were thus unable to combine resistance to aging (coming from E202Q or F338A substitutions) and enhanced reactivation (coming from D134H substitution) in the same double mutant hAChE protein.
In summary, our initial study shows that selected surface residue substitutions in the AChE molecule located remotely from the active center affect its catalytic parameters, OP inhibition and oxime reactivation and may be useful in designing oxime-hAChE couples for catalytic OP hydrolysis. However, combining diverse properties of single site mutations to form multiple site hAChE mutants does not result in a simple summation of ΔG values, rather there is a coupling of energy values that appear linked.