Hydrolysis of paraoxon

FIGURE 69.2. Reaction scheme of acetylcholinesterase inhibition, reactivation, and protection activities. OPH, 2-PAM, and paraoxon are used in the example. Reaction (I) - cholinesterase AChE reaction with ASCh Reaction (II) - inhibition of AChE inhibition by paraoxon Reaction (III) - aging of AChE associated with OP exposure Reaction (TV) -reactivation of AChE by 2-PAM Reaction (V) - hydrolysis of paraoxon by OPH Reaction (VI) - reaction of 2-PAM oxime with ASCh and Reaction (VII) - DTNB reaction with SCh. The indicates a photometrically detectable metabolite. 

As outlined in Figure 3, the hydrolysis of paraoxon by human serum A-esterase(s) is very similar to the phosphorylation of B-esterases, such as acetylcholinesterase, by paraoxon. Both reactions involve an initial binding of paraoxon to the enzyme, followed by a rapid conformational change that produces diethyl phosphate and p-nitrophenol from paraoxon. p-Nitrophenol is quickly released from the enzyme, leaving diethyl phosphate covalently bound to enzyme. At this point, A-esterase quickly releases diethyl phosphate as a result of interacting with a water molecule. However, B-esterases, such as acetylcholinesterase, retain the diethyl phosphate for a much longer period of time, thereby resulting in inhibition of the enzyme. 

While A-esterase(s) and B-esterases interact kinet-ically with paraoxon in a similar fashion (Figure 3), the molecular events occurring at their active sites during catalysis are probably very different. The active site of B-esterases such as acetylcholinesterase has been well characterized and contains a serine residue that is phosphorylated by paraoxon at the hydroxyl group. In contrast, the active site of A-est-erase(s) has not been studied as extensively, but it likely does not contain a serine residue that participates in the hydrolysis of paraoxon. Additionally, A-esterase(s) requires a divalent cation like calcium for activity, whereas B-esterases do not. 

The residue Asp-301, which is hydrogen bonded to His-254, has been proposed to deprotonate the bridging hydroxide nucleophile.253 Mutation of His-245 to Ala or Asp reduces the rate of hydrolysis of paraoxon by 1-2 orders of magnitude. In contrast, the hydrolysis for the slower substrate diethyl p-chlorophenyl phosphate increases by from 2- to 33-fold.253 This suggests that for the more activated... 

In human populations, serum PON1 exhibits a substrate-dependent polymorphism to the neurotoxic effects of organophosphates in those susceptible individuals that are deficient in PON1 (i.e., PM phenotype) (55). The PON1 catalyzes the hydrolysis of paraoxon, chlorpyrifos (Dursban), and other orgahophosphates. 

The hydrolysis of parathion proceeds slightly faster under acidic or neutral conditions of pH. The reverse is observed under alkaline conditions where the hydrolysis of paraoxon is approximately 7.5 times faster than parathion at pH 9.0 and 5.5 times faster at pH 10.4. 

Figure 6.3S Probing the hydrolysis of paraoxon by the organophosphorus (OPH)-modifled QDs. (Reproduced with permission from Ref. [202]. Copyright 2005 American Chemical Society).

Although the enzymatic hydrolysis reaction, in most cases, reduces the toxicity of OPs by converting them into less toxic metabolites, these metabolites also present a potential source of contamination to the environment. A very interesting mechanism of complete pesticide degradation was proposed by Mattozzi et al. describing the metabolic engineering of Pseudomonas putida strain to hydrolyze paraoxon and mineralize the hydrolysis products into sources of carbon and phosphorus. Previous studies reported that the hydrolysis of paraoxon to p-nitrophenol (PNP) and diethyl phosphate (DEP), by an OPH from Flavobacterium sp. strain ATCC 27551, rednces the toxicity of the pesticide 100-fold (Mattozzi et al. 2006). 

The hydrolysis of paraoxon (a P-O bond breakage) was monitored by incubating each cotton sample in five milliliters of 1 mM paraoxon in 20 mM CHES, pH 9.0, at 25°C with constant shaking at 150 rpm on a Lab-Line Junior Orbit Shaker. Aliquots were removed at various time points and the amount of p-nitrophenol released as a cleavage product was quantitated at 400 nm and converted to concentration using Beer s law and an extinction coefficient of 17,000 M cm ... 

FIGURE 59.2 (A) Hydrolysis of paraoxon with aryldiaUcylphos-phatase (paraoxonase, EC 3.1.8.1) and (B) hydrolysis of diisopropyl-fluorophosphate (DFP) with diisopropylfluorophosphatase (DFPase) (EC 3.1.82). 

---