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Measurement of the affinity and phosphorylation constants governing irreversible inhibition of cholinesterases by di-isopropyl phosphorofluoridate.

ABSTRACT: 1. A procedure is described for determining the affinity constant K(a) and the phosphorylation constant k(p) for the inhibition by di-isopropyl phosphorofluoridate of erythrocyte acetylcholinesterase and serum cholinesterase. The procedure depends on the use of a specially designed reaction vessel with which incubation times as short as 1.2sec. could be obtained at any convenient temperature. 2. The K(a) of acetylcholinesterase decreased from 1.58 (+/-0.22)x10(-3)m at 5 degrees to 1.17 (+/-0.10)x10(-3)m at 25 degrees and the associated change in enthalpy was 2980 cal. 3. The k(p) of acetylcholinesterase increased from 11.9 (+/-0.7)min.(-1) at 5 degrees to 40.7 (+/-1.4)min.(-1) at 25 degrees , indicating an activational energy of 9600 cal. The change in entropy associated with K(a) was 23.5 cal. degree(-1) at 25 degrees . 4. At 5 degrees , the K(a) and k(p) of serum cholinesterase were 9.95 (+/-1.10)x10(-6)m and 11.2 (+/-0.63)min.(-1) respectively. 5. The 150-fold difference in the inhibitory power of di-isopropyl phosphorofluoridate for the two cholinesterases was attributed entirely to differences in affinity.

SUBMITTER: Main AR

PROVIDER: S-EPMC1265167 | biostudies-other | 1966 Aug

REPOSITORIES: biostudies-other

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

Project description: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.

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