Phosphorylated AChE

CBs, like OPs, act as inhibitors of ChE. They are treated as substrates by the enzyme and carbamylate the serine of the active site (Figure 10.8). Speaking generally, car-bamylated AChE reactivates more rapidly than phosphorylated AChE. After aging has occurred, phosphorylation of the enzyme is effectively irreversible (see Section 10.2.4). Carbamylated AChE reactivates when preparations are diluted with water, a process that is accelerated in the presence of acetylcholine, which competes as a substrate. Thus, the measurement of AChE inhibition is complicated by the fact that reactivation occurs during the course of the assay. Carbamylated AChE is not reactivated by PAM and related compounds that are used as antidotes to OP poisoning (see Box 10.1). 

Organophosphates phosphorylate AChE by virtue of an electrophilic attack of the P atom on a serine hydroxyl of AChE, as shown in Eq. (21) for paraoxon (106) ... 

The formation of an unstable,covalent phosphoryl-AChE conjugate is evidently accompanied by a non-covalent reversible complex (Michaelis complex) between AChE and TDPI (AChE TDPI). Although TMPH (K. = 0.25 pM) was found to be 100 fold more powerful than TDPI (K = 0.02 mM) in terms of concentration required to achieve similar amount of Michaelis complex, TDPI provided better protection of AChE against irreversible phosphorylation by II. In this set of experiments, the efficiency of TDPI and TMPH were compared on the basis of equal concentration/affinity ratio were I/Kj = 10. 

As mentioned earlier, the phosphorylated AChE recovers very slowly. However, among organophosphates, the rate of recovery is relatively fast for dimethylphosphorylated AChE. For example, 50% recovery of a dimethylphosphorylated erythrocyte AChE is about 80 min as compared to 500 min for diethylphosphorylated AChE and almost negligible for isopropylphosphorylated AChE (Matsumura, 1985). Table 7.1 also shows a similar trend. 

In the case of organophosphate poisoning, the effectiveness of 2-PAM is dependent on its early administration. The phosphorylated AChE tends to convert to a form that cannot be reactivated by the oximes. This phenomenon is known as aging process. It involves the removal of an alkyl group from the dialkyl phosphorylated enzyme, producing an anionic phosphate moiety that is insensitive to oximes and other nucleophiles (Figure 7.17) (O Brien, 1976). 

The standard treatment of nerve agent-induced muscle toxicity calls for (1) reactivation of the phosphorylated AChE with an oxime, and (2) blockage of the nicotinic ACh receptor sites from the stimulating action of ACh with fii-tubocurarine. Oximes such as obidoxime, pralidoxime (2-pyridine aldoxime methochloride, 2-PAM) and a few others have been found very effective when given in combination with other drugs such as atropine, pretreatment with oximes varies with the ehemieal structures of the nerve agents and depends on the time after exposure. For example, it has been... 

Table 5 lists the half-lives of recovery for some OP-inhibited AChEs. In general OP-AChE complexes from dimethoxy-substituted OPs (e.g., malathion) spontaneously dephosphorylate faster than diethoxy (e.g., parathion) or diisopropoxy (e.g., DPP) complexes. Eto pointed out in 1974 that the stability of a phosphorylated AChE may be predicted from the stability of the specific OP inhibitor itself. One possibility is that methyl groups have less steric hindrance and greater electronegativity than ethyl or isopropyl groups. 

M blockers such as atropine (enters CNS), plus pralidoxime (2-PAM), may regenerate phosphorylated AChE, particularly at the skeletal neuromuscular junction (NMJ). Because time-dependent aging of the phosphorylated enzyme may decrease the effectiveness of the regenerator, 2-PAM should be used ASAP. 

In moderate or severe intoxication with an organophosphorus anti-ChE agent, the recommended adult dose of pralidoxime is 1—2 g, infused intravenously over not less than 5 minutes. If weakness is not relieved or if it recurs after 20-60 minutes, the dose should be repeated. Early treatment is very important to assure that the oxime reaches the phosphorylated AChE while the latter still can be reactivated. Many of the alkylphosphates are extremely lipid soluble, and if extensive partitioning into body fat has occurred and desulfuration is required for inhibition of AChE, toxicity will persist and symptoms may recur after initial treatment. With severe toxicities from the lipid-soluble agents, it is necessary to continue treatment with atropine and pralidoxime for a week or longer. 

Hobbiger, F. (1963). Reaclivatitm of phosphorylated AChE, Handbuch Exp. Pharmakol. IS, 921-988. 

Ah importaht differehce between irreversible phosphoester-derived AChEls ahd reversible AChEls is that the phosphorylated AChE cah uhdergo a process khowh as aging (Fig. 12.16). The aging process plays ah ... 

The phosphorylated AChE must be dephosphorylated back to liberate the enzyme AChE that could rapidly catalyze the... 

In organophosphates, two alkoxy groups are attached to the P atoms. When one of them is displaced by hydrolysis (nonenzymatic) any reactivation of the phosphorylated AChE does not occur and, therefore, the enzyme is irreversibly inhibited. The rate of displacement of such alkoxy group, known as aging is greater when an isopropoxy group is attached to the P atom. 

Various pharmacologic and therapeutic drugs have been tested to prevent or treat myopa y. Drugs effective in the treatment of neuromuscular signs of anti-ChE toxicity include (i) oximes that reactivate the phosphorylated AChE (Thiermann et al., 2005 Marrs and Vale, 2006), (ii) subparalyzing and paralyzing doses of rf-tubocura-rine (Clinton and Dettbam, 1987 Patterson et al., 1987),... 

---