Biotinylated covalent inhibitors of serine esterases allow detection and rapid isolation of neuropathy target esterase (NTE). This enzyme is the primary target site for those organophosphorus esters (OPs) which cause delayed neuropathy. One moiety of S9B (structurally similar to the active metabolite of TOCP) reacts specifically with the catalytic serine residue of NTE; the second moiety is a biotin molecule that allow the NTE-S9B adduct to be captured on avidin-Sepharose.
Title: Inhibition with spontaneous reactivation and the ongoing inhibition effect of esterases by biotinylated organophosphorus compounds: S9B as a model Estevez J, Barril J, Vilanova E Ref: Chemico-Biological Interactions, 187:397, 2010 : PubMed
The biotinylated organophosphorus compound 1-(saligenin cyclic phospho)-9-biotinyldiaminononane (S9B) has been used for the detection, labeling and isolation of the membrane-bound neuropathy target esterase (NTE) as it was considered a specific inhibitor of NTE. After incubation with the soluble fraction of chicken peripheral nerve, most of the soluble esterase activity was highly sensitive to S9B, indicating NTE-like esterases. A kinetic model equation was used to assume a multi-enzymatic system with three different simultaneously occurring molecular phenomena; (1) inhibition; (2) simultaneous spontaneous reactivation; and (3) ongoing inhibition (inhibition during the substrate reaction); to fit the data to analyze kinetic behavior. A high "ongoing inhibition" effect was observed in an enzymatic component. A three-dimensional fit of the model was applied. The best fitting model is compatible with three sensitive enzymatic entities (33, 52 and 15%), and only one spontaneously reactivate. The second-order rate constants of inhibition (k(i)=116 x 10(6), 4.6 x 10(6) and 0.28 x 10(6)M(-1)min(-1), respectively) and the spontaneous reactivation constant for the first sensitive component (k(r)=0.0054 min(-1)) were simultaneously estimated. These parameters are similar to those deduced in spontaneous reactivation experiments of the preinhibited samples with S9B. The estimated proportions of enzymatic components are similar to those previously observed in inhibition experiments with mipafox, demonstrating that this kinetic approach offers consistent results.
Title: Neuropathy target esterase and a homologous Drosophila neurodegeneration-associated mutant protein contain a novel domain conserved from bacteria to man Lush MJ, Li Y, Read DJ, Willis AC, Glynn P Ref: Biochemical Journal, 332 ( Pt 1):1, 1998 : PubMed
The N-terminal amino acid sequences of proteolytic fragments of neuropathy target esterase (NTE), covalently labelled on its active-site serine by a biotinylated organophosphorus ester, were determined and used to deduce the location of this serine residue and to initiate cloning of its cDNA. A putative NTE clone, isolated from a human foetal brain cDNA library, encoded a 1327 residue polypeptide with no homology to any known serine esterases or proteases. The active-site serine of NTE (Ser-966) lay in the centre of a predicted hydrophobic helix within a 200-amino-acid C-terminal domain with marked similarity to conceptual proteins in bacteria, yeast and nematodes; these proteins may comprise a novel family of potential serine hydrolases. The Swiss Cheese protein which, when mutated, leads to widespread cell death in Drosophila brain [Kretzschmar, Hasan, Sharma, Heisenberg and Benzer (1997) J. Neurosci. 17, 7425-7432], was strikingly homologous to NTE, suggesting that genetically altered NTE may be involved in human neurodegenerative disease.
Title: Synthesis and characterization of a biotinylated organophosphorus ester for detection and affinity purification of a brain serine esterase: neuropathy target esterase Glynn P, Read DJ, Guo R, Wylie S, Johnson MK Ref: Biochemical Journal, 301 ( Pt 2):551, 1994 : PubMed
We have synthesized a novel stable precursor, saligenin phosphorotrichloridate, which, on reaction with N-monobiotinyldiamines, generates a series of biotinylated covalent inhibitors of serine esterases. A homologue designated S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane] was selected to allow detection and rapid isolation of neuropathy target esterase (NTE). This enzyme is the primary target site for those organophosphorus esters (OPs) which cause delayed neuropathy. NTE comprises about 0.03% of the total protein in brain microsomal fractions and has resisted purification attempts over many years. S9B is a potent progressive inhibitor of NTE esteratic activity (second-order rate constant 1.4 x 10(7) M-1.min-1). Incubation of S9B with brain microsomes led to specific covalent labelling of NTE as determined by detection of a biotinylated 155 kDa polypeptide on Western blots. Specificity of S9B labelling was further demonstrated by inhibition with the neuropathic OP mipafox. Biotinyl-NTE in SDS-solubilized S9B-labelled microsomes was adsorbed on to avidin-Sepharose and subsequently eluted, yielding a fraction enriched approx. 1000-fold in NTE by a single step with recoveries of 30%. Essentially pure NTE was obtained after separation from two endogenous biotinylated polypeptides (120 and 70 kDa) in avidin-Sepharose eluates by preparative SDS/PAGE. Other biotinylated saligenin phosphoramidates derived from the same precursor may be useful for detection and isolation of other serine esterases and proteinases.
