758 15. EFFECTS OF VARIOUS FACTORS ON INHIBITION 



with the temperature. This enthalpy of activation is related to the Arrhe- 

 nius activation energy as follows: E^^^ = AH* + RT. If AH* changes 

 with temperature, the plot will not be linear and the slope at any point 

 will give the AH* at that temperature. The entropy of activation may be 

 obtained from the intercept on the log (JcIT) axis, which is equal to log {Rj 

 Nh) + {AS*I2.30^R). 



These concepts may be applied to any rate constants, not only for the 

 formation of the EI complex, but also, for example, to A;_i for the dissocia- 

 tion of the EI complex, or to k.^ for the dissociation of the ES complex into 

 products. The enthalpies and entropies of activation are actually known for 

 many enzyme reactions, both for the formation and the dissociation of the 

 ES complexes (see Lumry, 1959, Laidler, 1958), but very little has been 

 reported on rate constants related to enzyme inhibition. Information on 

 the course of the binding reaction could be obtained from temperature 

 studies on the rates at which inhibitions develop. As Laidler (1958) points 

 out, an entropy change upon formation of EI* could result from either 

 structural changes in the protein at the active center or in alterations of 

 the state of hydration. If the enzyme unfolds during the formation of the 

 activated complex, J*S'* will be positive, and there is some indirect evidence 

 that such may occur in the formation of the ES* activated complex. How- 

 ever, 'the entropy changes due to modifications in the hydration may 

 often mask these structural effects. If charges on the enzyme and inhibitor 

 are neutralized during the complex formation, water will be set free and 

 AS* will be positive. Unfortunately the rates of inhibition in many im- 

 portant cases are too rapid to measure with the techniques that are available 

 and hence the data from which activation energies might be calculated 

 are not available. 



More temperature studies on inhibition rates have been done using 

 cholinesterase than any other enzyme, but the extent and accuracy of the 

 data are often insufficient for thermodynamic characterization. The rate 

 of inhibition of human plasma cholinesterase by HgClg is markedly de- 

 pendent on the temperature (Goldstein and Doherty, 1951). For example, 

 a concentration of 0.114 niM inhibited 76.4% after 4 h at 37°, but at 0" 

 there was only 18.5% inhibition after 24 h and 63.7% inhibition after 7 

 days. The inactivation rate at 37° was about a hundred times greater than 

 at 0°. The kinetics of the inhibition led to the postulate that an initial 

 inhibition (perhaps as the result of reaction with SH groups) is followed 

 by an irreversible inactivation. The temperature coefficient is much higher 

 for the secondary inactivation than for the early inhibition, indicating that 

 the former is akin to a denaturation process. The inhibitions of human 

 erythrocyte cholinesterase by urethane (Shukuya, 1953) and salicylate 

 (Shukuya, 1954) also seem to involve two types of inhibition mechanisms, 

 since the temperature coefficients were high when high concentrations of 

 the inhibitors were used, implying a denaturation-type inactivation, and 



