The relevance of inhibitor-substrate interactions when measuring neuropathy target esterase inhibition

Neuropathy target esterase (NTE), thought to be the target for organophosph ate polyneuropathy, is operationally defined as that neural phenyl valerate esterase resistant to paraoxon (40 mu M) and sensitive to mipafox (50 mu M ; 20 min, pH 8.0, 37 degrees C). The time course of inhibition of particula te paraoxon pretreated esterases by mipafox showed that the lines indicatin g the rate of inhibition did not pass through the log 100% activity when ex trapolated at zero time. Slopes of inhibition of NTE were not linearly rela ted to the concentration of mipafox. Kinetic parameters derived from Wilkin son type plots were: K-a=49-199 mu M, k(+2)=0.24-0.64 min(-1) and k(a)=3.1- 5.0 mM(-1) m(-1) When mipafox was removed (either by dilution or centrifuga tion) before the addition of phenyl valerate intercepts below 100% disappea red. We confirm that the formation of Michaelis complex between NTE and mip afox is not prevented by phenyl valerate and that inhibition proceeds after addition of phenyl valerate. We compared inhibitions obtained with experim ents by using the traditional method (sequential incubation with inhibitors and phenyl valerate) to those obtained with a method where mipafox is remo ved before the addition of substrate. When calculating fixed-time 50% inhib itory concentrations (IC(50)s) of some inhibitors for NTE, the longer the h ydrolysis time, the lower were the IC(50)s. Therefore, the inhibitory poten cy of certain NTE inhibitors, is accurately assessed only when calculating second-order rate constants (k(a)).