Dataset Information

Rapid aging of neurotoxic esterase after inhibition by di-isopropyl phosphorofluoridate.

ABSTRACT: 1. It was proposed [Johnson (1974) J. Neurochem.23, 785-789] that an essential step in the genesis of delayed neuropathy caused by some organophosphorus esters was aging of phosphorylated neurotoxic esterase, involving generation of a charged monosubstituted phosphoric acid residue on the protein. 2. Neurotoxic esterase of hen brain was inhibited with di-isopropyl phosphorofluoridate either unlabelled or mixed-labelled with (3)H and (32)P. 3. Reactivation of inhibited enzyme by KF was possible only immediately after a brief inhibition:aging at pH8.0 and 37 degrees C occurred with a half-life of about 2-4min. 4. When the radiolabelled enzyme was studied no loss of label was observed during the expected aging period, but a change in the nature of the bound radioisotopes occurred (half-life=3.25min). 5. Alkaline hydrolysis of labelled enzyme liberated di-isopropyl phosphate at early times after labelling, but increasing amounts of monoisopropyl phosphate plus a volatile tritiated compound (possibly propan-2-ol) at later times. 6. Treatment of labelled enzyme with KF released di-isopropyl phosphate and caused reactivation of enzyme to similar degrees. It is concluded that the chemical change from di-isopropyl phosphoryl-enzyme to mono-isopropyl phosphoryl-enzyme and the loss of reactivatibility are related. 7. The rate of aging is similar at pH5.2, 6.5 and 8. Aging is unaffected by addition of reduced glutathione and imidazole at pH5.2 or 8, and none of the transferred (3)H is trapped by these reagents. The mechanism of aging must be different from the better-known dealkylation aging of the cholinesterases.

SUBMITTER: Clothier B

PROVIDER: S-EPMC1186405 | biostudies-other | 1979 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Neurotoxic esterase activity was measured in homogenates of human placenta and hen brain, spinal cord, liver, kidney and spleen. The activity in liver comprised less than 20% of the Paraoxon-resistant esterases, but in the other tissues neurotoxic esterase accounted for over 50%. The same tissues were labelled with [3H]di-isopropyl phosphorofluoridate, and any isopropyl group transferred on to protein during 'aging' of the labelled enzymes (alkali-volatilizable tritium) was measured. No Paraoxon-sensitive labelled sites were found to age in this way in any tissue. In brain, the Paraoxon-resistant alkali-volatilizable-tritium-labelled sites correlated with the number of neurotoxic esterase labelled sites, indicating that 'aging' and isopropyl group transfer were 100% efficient. The site receiving the transferred isopropyl group was characterized by analysing the distribution of radiolabelled proteins on gel-filtration chromatography in the presence of SDS. In particulate preparations from each tissue, the protein-bound alkali-volatilizable tritium (transferred isopropyl group) was attached to a polypeptide of Mr 178 000. This same polypeptide also bore the isopropyl-phosphoryl group of neurotoxic esterase, indicating that aging of neurotoxic esterase is an intramolecular group transfer. The apparent turnover number for the enzyme (average 1.6 X 10(5) min-1) was approximately the same in each hen tissue, confirming that closely similar enzymes were present in brain, spinal cord, liver and spleen. The apparent turnover for the human enzyme was 1.8-fold higher than that for the hen enzyme. The concentration of the neurotoxic esterase phosphorylated subunit in brain, spinal cord, spleen, placenta and liver was 14.6, 3.8, 7.4, 3.3 and 3.8 pmol/g of tissue. The evidence indicated that neurotoxic esterase is present in each tissue except kidney, and that isopropyl group transfer on 'aging' occurs on this enzyme only. This process is an intramolecular transfer of the group within the same polypeptide.

Project description:Organophosphate-inhibited cholinesterases can be reactivated by nucleophilic compounds. Sometimes phosphylated (phosphorylated or phosphonylated) cholinesterases become progressively refractory to reactivation; this can result from different reactions. The most frequent process, termed 'aging', involves the dealkylation of an alkoxy group on the phosphyl moiety through a carbocation mechanism. In attempting to determine the amino acid residues involved in the aging of butyrylcholinesterase (BuChE), the human BuChE gene was mutated at several positions corresponding to residues located at the rim of the active site gorge and in the vicinity of the active site. Mutant enzymes were expressed in Chinese hamster ovary cells. Wild-type BuChE and mutants were inhibited by di-isopropylfluorophosphate at pH 8.0 and 25 degrees C. Di-isopropyl-phosphorylated enzymes were incubated with the nucleophilic oxime 2-pyridine aldoxime methiodide and their reactivatability was determined. Reactivatability was expressed by the first-order rate constant of aging and/or the half-life of aging (t12). The t12 was found to be of the order of 60 min for wild-type BuChE. Mutations on Glu-197 increased t12 60-fold. Mutation W82A increased t12 13-fold. Mutation D70G increased t12 8-fold. Mutations in the vicinity of the active site serine residue had either moderate or no effect on aging; t12 was doubled for F329C and F329A, increased only 4-fold for the double mutant A328G+F329S, and no change was observed for the A328G mutant, indicating that the isopropoxy chain to be dealkylated does not directly interact with Ala-328 and Phe-329. These results were interpreted by molecular modelling of di-isopropylphosphorylated wild-type and mutant enzymes. Molecular dynamics simulations indicated that the isopropyl chain that is lost interacted with Trp-82, suggesting that Trp-82 has a role in stabilizing the carbonium ion that is released in the dealkylation step. This study emphasized the important role of the Glu-197 carboxylate in stabilizing the developing carbocation, and the allosteric control of the dealkylation reaction by Asp-70. Indeed, although Asp-70 does not interact with the phosphoryl moiety, mutation D70G affects the rate of aging. This indirect control was interpreted in terms of change in the conformational state of Trp-82 owing to internal motions of the Omega loop (Cys-65-Cys-92) in the mutant enzyme.

Dataset Information

Rapid aging of neurotoxic esterase after inhibition by di-isopropyl phosphorofluoridate.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets