KATES OF INHIBITION OF PURE ENZYMES 561 



The rate constant is determined by the free energy of activation, Ji^*, 

 and the enthalpy of activation derived from the plotting procedure is only 

 a ]3art of this, the other part involving the entropy of activation, J»S*. 

 The relative contril^ntions of the enthalpy and entropy to the free energy 

 of activation will vary with the nature of the inhibition. The entropy term 

 may well be quite appreciable, because not only will the deformation of the 

 inhibitor molecule in the activated complex be a factor, but inasmuch as 

 many inhibitors are ionic, the displacement of water molecules from the ionic 

 groups of both inhibitor and enzyme during the formation of the complex 

 may involve large changes in entropy. Evaluation of the exjD (J>S*/i?) 

 term in Eq. 12-39 leads to the following conclusions about the entropy 

 effect: an increase in entropy of 3 cal/mole/deg will increase k^ by a factor 

 of 4.53, an increase of 5 cal/mole/deg by a factor of 12.3, an increase of 10 

 cal/mole/deg by a factor of 151, and an increase of 20 cal/mole/deg by a 

 factor of 22,100. Thus the entro])y change may be a very important factor 

 in determining the rate of inhiliition. Some idea of the magnitude of J*S* 

 may be obtained from the values of k^ and JH* determined independently. 

 Such data appear to be available for only one inhibition, the inactivation 

 of liver monoamine oxidase by iproniazid. Davison (1957) found the rate 

 constant, k^, to be 1.65x10^ liters mole~^ min~^ at 37^ and the enthalpy 

 of activation to be about 30 kcal/mole. Substituting these values in Eq. 

 12-39. J>S* is found to be 44.4 cal/mole/deg, which is surprisingly high. If 

 it were not for this great increase in entropy, the reaction of iproniazid 

 with the monoamine oxidase would be extremely slow. Actually, the free 

 energy of activation, AF*, is only 16.2 kcal/mole and the entropy change 

 may be shown to increase the rate by a factor of 4.7 X 10^. Such a high value 

 for AS* might indicate a change in the enzyme structure upon inactivation, 

 i.e., a partial denaturation. 



When the free energy of activation for the binding of an inhibitor to 

 an enzyme is below 5 kcal/mole, it is likely that the process is limited by 

 diffusion and that almost every collision of an inhibitor molecule with the 

 reactive site on the enzyme leads to binding. Many ionic reactions, such 

 as the inhibition of succinic dehydrogenase by malonate, probably fall 

 in this category. It is possible that such low free energies of activation are 

 the result of entropy increases attending the rearrangement of hydration 

 water. 



Secondary Inactivation of Enzymes during Inhibition 



An enzyme is frequently unstable under the conditions used to determine 

 inhibition. It is well known that many enzymes when extracted from cells 

 and subjected to the abnormal media used to study their activity gradually 

 lose this activity, particularly at 37-38''. This inactivation may be a de- 

 naturation of the enzyme protein or it may relate to the loss of some bound 

 cofactor. Determination of the time course of an inhibition must take into 



