RATES OF INHIBITION OF PURE ENZYMES 569 



The course of the true inhibition is plotted as curve 2 in Fig. 12-20 for the 

 case in which A'g = 5^*0. The true inhibition is reduced in comparison to 

 curve 3 because the EI complex is inactivated at a rather rapid rate and 

 (EI) does not attain such high levels as when no inactivation occurs. 



The Enzyme Is Inhibited and Reactivated Simultaneously 



Certain inhibitors, notably the organophosphorus compounds, bind to 

 the active center and then react chemically with enzyme to inactivate. In 

 some cases the inactivated enzyme may be reactivated by a chemical reac- 

 tion involving the splitting off of the attached residue. The kinetics for 

 such an inhibition have been treated by van Asperen and Dekuijzen (1958) 

 for the cholinesterases of houseflies and mouse brain. The following reac- 

 tions occur: 



E + I — EI K, = (E)(I)/(EI) 



El -> EP + V V, = Ai(El) 



EP ~> E -r P i'2 = A:2(EP) 



where the inhibitor is 0.0-dimethyl-0-2.2-dichlorovinyl phosphate (DDVP), 

 P is dimethyl phosphate and V is dichlorovinyl alcohol, P and V being the 

 products formed from this particular inhibitor. The phosphorylated en- 

 zyme EP is inactive whereas EI, although inactive, may be considered as 

 active since it can give rise to active enzyme. When v., = 0, the final in- 

 hibition will depend only on the relative concentrations or amounts of the 

 enzyme and the inhibitor, but when v.^ is finite, the inhibition will rise to 

 a level which may remain constant, a steady state in which the formation 

 and breakdown of EP are equal and (E) is constant. Steady inhibition thus 

 occurs when v^ — Vo. Curves are presented for various concentraions of 

 DDVP and it was found that it requires around 30 to 60 min for a steady 

 state to be reached (Fig. 12-22). 



The potency of an inhibitor in this situation depends on the rate at 

 which EP is dissociated, i.e., the rate of reactivation, the greater this rate, 

 the higher the concentration of an inhibitor necessary to produce a chosen 

 final level of inhibition. The tw^o cholinesterases from flies and mouse 

 brain differ by a factor of around 50 in their resistance to DDVP because 

 of different rates of reactivation. 



Preincubation Procedure and Determination of the Component with 

 Which the Inhibitor Reacts 



When the inhibitor and all the components of the enzyme system are 

 mixed simultaneously, or when the inhibitor is added while the enzyme 

 reaction is proceeding, the situation is generally more difficult to deal with 



