Acetylcholinesterase formation

Enzyme-Catalyzed Reactions Enzymes are highly specific catalysts for biochemical reactions, with each enzyme showing a selectivity for a single reactant, or substrate. For example, acetylcholinesterase is an enzyme that catalyzes the decomposition of the neurotransmitter acetylcholine to choline and acetic acid. Many enzyme-substrate reactions follow a simple mechanism consisting of the initial formation of an enzyme-substrate complex, ES, which subsequently decomposes to form product, releasing the enzyme to react again. 

Acetylcholine is formed from acetyl CoA (produced as a byproduct of the citric acid and glycolytic pathways) and choline (component of membrane lipids) by the enzyme choline acetyltransferase (ChAT). Following release it is degraded in the extracellular space by the enzyme acetylcholinesterase (AChE) to acetate and choline. The formation of acetylcholine is limited by the intracellular concentration of choline, which is determined by the (re)uptake of choline into the nerve ending (Taylor Brown, 1994). 

The postsynaptic membrane opposite release sites is also highly specialized, consisting of folds of plasma membrane containing a high density of nicotinic ACh receptors (nAChRs). Basal lamina matrix proteins are important for the formation and maintenance of the NMJ and are concentrated in the cleft. Acetylcholinesterase (AChE), an enzyme that hydrolyzes ACh to acetate and choline to inactivate the neurotransmitter, is associated with the basal lamina (see Ch. 11). 

Both nicotinic receptors and acetylcholinesterase are regulated tightly during differentiation and synapse formation 202... 

Fish rapidly absorb, metabolize, and excrete chlorpyrifos from the diet (Barron etal. 1991). The mechanism of action of chlorpyrifos occurs via phosphorylation of the active site of acetylcholinesterase after initial formation of chlorpyrifos oxon by oxidative desulfuration. In studies with channel catfish (Ictalurus punctatus), the oral bioavailability of chlorpyrifos was 41%, substantially higher than in mammals. Catfish muscle contained less than 5% of the oral dose with an... 

More recently, B. Goldstein and McDonagh demonstrated that the native protein fluorescence (280-nm excitation, 330-nm emission) of red-cell membranes exposed in vitro to ozone at 1 ppm was a somewhat more sensitive indicator of ozone effect than other characteristics measured in the same em, including oxidation of cell-membrane sulfhydiyl groups, loss of acetylcholinesterase activity, and formation of lipid peroxide breakdown products. 

Another drug with a high incidence of hepatotoxicity is the acetylcholinesterase inhibitor tacrine. Binding of reactive metabolites to liver tissue correlated with the formation of a 7-hydroxy metabolite [13], highly suggestive of a quinone imine metabolite as the reactive species. Such a metabolite would be formed by further oxidation of 7-hydroxy tacrine (Figure 8.11). 

The first suggestion of a practical form of antidotal therapy came in 1949 from Hestrin, who found that acetylcholinesterase (AChE) catalyzed the formation of acetohydroxamlc acid when incubated with sodium acetate and hydroxylamine. Critical in vitro studies in the next decade led to the development of a practical approach to therapy. The crucial concept in these studies was the recognition that the compound formed when AChE reacted with a phosphorus ester was a covalent phosphoryl-enzyme Intermediate similar to that formed in the hydrolysis of acetylcholine. 3 Wilson and colleagues, beginning in 1951, demonstrated that AChE inhibited by alkyl phosphate esters (tetraethyl pyrophosphate, TEPP) could be reactivated by water, but that free enzyme formed much more rapidly in the presence of hydroxylamine. 0 21 Similar results... 

Fig. 3. Stereospecific hydrogen bond formation of phenyl N-methylcarbamates with acetylcholinesterase 141 (reproduced with permission from Academic Press, Inc.)...

TK interactions between metals and organic compounds are also possible phenan-threne appears to enhance the uptake of cadmium from sediment in the amphipod Hyalella azteca (Gust and Fleeger 2005). In the same species, chlorpyrifos enhances the accumulation of methyl mercury, but methyl mercury reduces acetylcholinesterase inhibition caused by chlorpyrifos, presumably due to the formation of a chlor-pyrifos-MeHg complex (Steevens and Benson 1999). 

Challenging the dynamic thiolester system CDS-1B with acetylcholinesterase resulted in similar product formations, but a slightly slower enzyme resolution process, than CDS-1A due to the larger size of CDS-1B. To probe the effect of the thiol moiety, dynamic thiolester systems, using only one thiol per system, were furthermore generated and applied to the enzyme resolution process. Thus, the additional CDS-1B, 1C, ID, and IE were prepared with equimolar amounts of five thiolesters 1A-E and one equivalent of either thiol 2, 8,10, or 12 (Table 1). These systems contained ten thiolesters and two thiols. Challenging these systems with acetylcholinesterase resulted in half-lives of formation of the different hydrolysis products, acetic, propionic, and butyric acids, as shown in Table 1. These results indicate that only CDS-1 A (Table 1, Entry 2), generated from five thiolesters 1A-E... 

Dynamic thiolester systems were generated from one equivalent of each thiolesters 1A-E and additional thiol in NaOD/DaPfXi buffer solution at pD 7.0 and then applied to acetylcholinesterase resolution. The rates of product formations were followed by 1H-NMR spectroscopy... 

Enzyme electrodes for choline and acetylcholine (300, 301) and for the analysis of choline-containing phospholipids (303, 304) is obtained by immobilizing choline oxidase or choline oxidase and acetylcholinesterase on membranes at the tip of platinum electrodes. The formation of hydrogen peroxide is monitored ... 

Antibodies to OP-tyrosine will be made. These antibodies will be used to diagnose OP exposure in a biosensor assay with saliva, sweat, or urine. New biomarkers of OP exposure will be identified using mass spectrometry and the new OP-tyrosine antibodies. The identification of new biomarkers for low-dose OP exposme is expected to lead to an understanding of how neurotoxicity is caused by OP doses that are too low to inhibit acetylcholinesterase. For example, it is possible that disruption of microtubule polymerization by OP-adduct formation may explain cognitive impairment from OP exposure. 

FIGURE 65.2. Reactivation of phosphorylated acetylcholinesterase with pralidoxime and formation of reactivated enzyme and phosphorylated oxime. 

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