636 13. REVERSAL OF INHIBITION 



perature, (e) the pH, and (/) the ionic strength of the medium. The effect 

 of temperature is usually quite marked and the apparent activation 

 energies are high. Until the details of the kinetics can be worked out, it 

 will not be possible to attribute these activation energies to any partic- 

 ular step in the reactivation. The entropy of activation for the reactiva- 

 tion of TEPP-inhibited cholinesterase by hydroxylamine is — 37 cal/ 

 mole/deg and thus is very high (Wilson, 1952b). This was interpreted as 

 indicating a very restricted geometric path of attack although other fac- 

 tors may certainly contribute to this. A plot of the reactivation rate against 

 pH shows in every case studied a rather sharp optimal pH, which varies 

 with the enzyme, inhibitor, and reactivator used and is usually between 

 6.9 and 8.3 (Wilson et al., 1955; Cunningham, 1954; Davies and Green, 

 1956). The effects of pH and ionic strength on the reactivation rate are 

 perhaps partially explained on the basis of the electrostatic interactions 

 between the reactivator and the anionic site and partially by the changes 

 induced in the histidine groups that are phosphorylated at the active 

 site (see Chapter 14). 



It is interesting in connection with what was said previously about 

 secondary reactions that proceed during inhibition and reduce the reversibil- 

 ity, that the longer an enzyme is incubated with an organopohsphorus inhi- 

 bitor, the more irreversible becomes the inhibition. This may be due to the 

 transfer of the phosphoryl group from the primary site of attack, which is 

 probably histidine, to an adjacent serine (Davies and Green. 1956). Reacti- 

 vation does not occur when the enzyme has reached this final irreversible 

 stage (Sanderson and Edson, 1959). This perhaps explains why the survival 

 of rats poisoned by organophosphorus compounds and treated with reac- 

 tivators is dependent on the time at which the reactivator is given. If 

 the reactivator is administered within 1 hr following the poisoning, the 

 survival is down to 10% (Fleisher et al., 1960). 



The interest in this type of reactivation is justified not only by the im- 

 portant information it provides on the enzyme active site and the forces 

 and steric factors involved, but also by the practical significance such reac- 

 tivators have in the treatment of poisoning by the organophosphorus com- 

 pounds. It has been shown that 2-PAM and related reactivators are quite 

 effective in protecting animals against these inhibitors and efforts are being 

 made to develop reactivators that are distributed in the body in a satisfac- 

 tory manner to bring about restoration of cholinesterase activity in the 

 critical tissues. This aspect of the jjroblem will be discussed in a later 

 chapter on cholinesterase inhibitors (Volume 3). Although the inhibition 

 by organoi^hosphorus compounds is the only known example of such reac- 

 tivation, it may be speculated that other cases will soon ]}e demonstrated, 

 especially in the field of monoamine oxidase inhibitors, since they too pre- 

 sumably react chemically with the active site in a manner related to the 

 amine substrates. 



