Neuropathy Target Esterase Biosensor

Abstract

Neuropathy Target Esterase (NTE), is a membrane-bound protein found in neurons of vertebrates (Glynn, 1999; Atkins & Glynn, 2000; van Tienhoven et al., 2002; Li et al., 2003; Makhaeva et al., 2003; Kropp et al., 2004), has been shown to be necessary for embryonic development in mice, and is believed to be involved in cell-signaling pathways and lipid trafficking (Glynn, 1999). NTE has serine esterase activity and can hydrolyze ester, peptide, and amide bonds. The nucleophilic serine residue (active site) of NTE attacks the carbonyl carbon atom of the substrate, forming a covalent acyl-enzyme intermediate, which is subsequently hydrolyzed. A consequence of this reaction mechanism is that the esterase activity of NTE is susceptible to covalent inhibition by organophosphorus esters (OPs) with which it forms an analogous phosphyl-enzyme intermediate. Irreversible binding of some OP compounds to the active serine site results in a debilitating neural disease known as OP-induced delayed neuropathy (OPIDN) (Glynn, 1999). Signs of OPIDN include flaccid paralysis of the lower limbs, which becomes evident two to three weeks after exposure to neuropathic OPs. Recovery from this disease is usually poor, and there is no specific treatment. In addition, mutations in the NTE gene have been linked to motor neuron disease (Rainier et al., 2008). Because NTE plays a central role in both chemically induced and spontaneously occurring neurological diseases, approaches that can help measure its esterase activity and inhibition are of tremendous scientific and commercial importance. Because NTE is difficult to produce for research purposes, research to study its esterase activity is typically done using a fragment of the NTE protein that contains the esterase activity and can be more easily produced. One such fragment, known as NEST, (Atkins & Glynn, 2000; Forshaw et al., 2001; Kropp et al., 2004) reacts with esters and inhibitors in a manner very similar to NTE. Conventionally, the esterase activity of NTE (or NEST) is measured using two distinct steps. In the first step, a solution containing phenyl valerate is brought into contact with NEST or NTE protein solution, whose esterase activity reacts with a portion of the artificial substrate phenyl valerate to form phenol. In the second step, the concentration of phenol in the solution is determined either colorimetrically, in the presence of 4-amino antipyrine