Mizrahi D

References (3)

Title : The role of AChE active site gorge in determining stereoselectivity of charged and noncharged VX enantiomers - Ordentlich_2005_Chem.Biol.Interact_157-158_191
Author(s) : Ordentlich A , Barak D , Sod-Moriah G , Kaplan D , Mizrahi D , Segall Y , Kronman C , Karton Y , Lazar A , Marcus D , Velan B , Shafferman A
Ref : Chemico-Biological Interactions , 157-158 :191 , 2005
Abstract : The reactivity of human acetylcholinesterase (HuAChE) toward the chemical warfare agent VX [O-ethyl S-[2-(diisopropylamino)ethyl] methyl-phosphonothioate] and its stereoselectivity toward the P(S)-enantiomer were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere nc-VX [O-ethyl S-(3-isopropyl-4-methyl-pentyl) methylphosphonothioate]. Stereoselectivity of the wild-type HuAChE toward VX(S) is manifested by a 115-fold higher bimolecular rate constant (1.4 x 10(8) min(-1) M(-1)) as compared to that of VX(R). HuAChE was also 12,500-fold more reactive toward VX(S) than toward nc-VX(S), demonstrating the significance of the polar interactions of the ammonium substituent to their overall affinity toward VX. Indeed, substitution of the cation-binding subsite residue Trp86 by alanine resulted in a decrease of three orders of magnitude in HuAChE reactivity toward both VX enantiomers, with only a marginal effect on the reactivity toward the enantiomers of nc-VX. These results demonstrate that accommodation of the charged moieties of both VX enantiomers depends predominantly on interactions with the aromatic moiety of Trp86. Yet, these interactions seem to limit the stereoselectivity toward the P(S)-enantiomer, which for charged methylphosphonates is much lower than for the noncharged analogs, like sarin or soman. Marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 by asparagine (D74N) practically abolished stereoselectivity toward VX(S) (a 130-fold decrease), while substitution which retained the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers originates predominantly from a different orientation of the charged leaving group with respect to residue Asp74. Such different orientations of the charged leaving group in the HuAChE adducts of the VX enantiomers seem to be a consequence of intramolecular interactions with the bulky phosphorus alkoxy group.
ESTHER : Ordentlich_2005_Chem.Biol.Interact_157-158_191
PubMedSearch : Ordentlich_2005_Chem.Biol.Interact_157-158_191
PubMedID: 16289014

Title : Stereoselectivity toward VX is determined by interactions with residues of the acyl pocket as well as of the peripheral anionic site of AChE - Ordentlich_2004_Biochemistry_43_11255
Author(s) : Ordentlich A , Barak D , Sod-Moriah G , Kaplan D , Mizrahi D , Segall Y , Kronman C , Karton Y , Lazar A , Marcus D , Velan B , Shafferman A
Ref : Biochemistry , 43 :11255 , 2004
Abstract : The origins of human acetylcholinesterase (HuAChE) reactivity toward the lethal chemical warfare agent O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) and its stereoselectivity toward the P(S)-VX enantiomer (VX(S)) were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere O-ethyl S-(3-isopropyl-4-methylpentyl) methylphosphonothioate (nc-VX) as well as echothiophate and its noncharged analogue. Reactivity of wild-type HuAChE toward VX(S) was 115-fold higher than that toward VX(R), with bimolecular rate constants of 1.4 x 10(8) and 1.2 x 10(6) min(-1) M(-1). HuAChE was also 12500-fold more reactive toward VX(S) than toward nc-VX(S). Substitution of the cation binding subsite residue Trp86 with alanine resulted in a 3 order of magnitude decrease in HuAChE reactivity toward both VX enantiomers, while this replacement had an only marginal effect on the reactivity toward the enantiomers of nc-VX and the noncharged echothiophate. These results attest to the critical role played by Trp86 in accommodating the charged moieties of both VX enantiomers. A marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 with asparagine (D74N) practically abolished stereoselectivity toward VX(S) (130-fold decrease), while a substitution which retains the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers is mainly a result of a different interaction of the charged leaving group with Asp74.
ESTHER : Ordentlich_2004_Biochemistry_43_11255
PubMedSearch : Ordentlich_2004_Biochemistry_43_11255
PubMedID: 15366935

Title : Evidence for P-N bond scission in phosphoroamidate nerve agent adducts of human acetylcholinesterase - Barak_2000_Biochemistry_39_1156
Author(s) : Barak D , Ordentlich A , Kaplan D , Barak R , Mizrahi D , Kronman C , Segall Y , Velan B , Shafferman A
Ref : Biochemistry , 39 :1156 , 2000
Abstract : Acetylcholinesterases (AChEs) form conjugates with certain highly toxic organophosphorus (OP) agents that become gradually resistant to reactivation. This phenomenon termed "aging" is a major factor limiting the effectiveness of therapy in certain cases of OP poisoning. While AChE adducts with phosphonates and phosphates are known to age through scission of the alkoxy C-O bond, the aging path for adducts with phosphoroamidates (P-N agents) like the nerve agent N,N-dimethylphosphonocyanoamidate (tabun) is not clear. Here we report that conjugates of tabun and of its butyl analogue (butyl-tabun) with the E202Q and F338A human AChEs (HuAChEs) age at similar rates to that of the wild-type enzyme. This is in marked contrast to the large effect of these substitutions on the aging of corresponding adducts with phosphates and phosphonates, suggesting that a different aging mechanism may be involved. Both tabun and butyl-tabun appear to be similarly accommodated in the active center, as suggested by molecular modeling and by kinetic studies of phosphylation and aging with a series of HuAChE mutants (E202Q, F338A, F295A, F297A, and F295L/F297V). Mass spectrometric analysis shows that HuAChE adduct formation with tabun and butyl-tabun occurs through loss of cyanide and that during the aging process both of these adducts show a mass decrease of 28 +/- 4 Da. Due to the nature of the alkoxy substituent, such mass decrease can be unequivocally assigned to loss of the dimethylamino group, at least for the butyl-tabun conjugate. This is the first demonstration that AChE adducts with toxic P-N agents can undergo aging through scission of the P-N bond.
ESTHER : Barak_2000_Biochemistry_39_1156
PubMedSearch : Barak_2000_Biochemistry_39_1156
PubMedID: 10653663