RATES OF INHIBITION OF PUEE ENZYMES 563 



because the determination of rate and inhibition constants, or even of the 

 type of inhibition, may otherwise be seriously in error. 



The mechanisms by which the stabiKty of an enzyme can be affected by 

 the binding of an inhibitor are generally unknown but some suggestions 

 may be made. The secondary and tertiary structures of enzyme proteins are 

 probably often sensitive to the ionic charge distribution on the enzyme 

 surface. The neutralization of such ionic groups or the introduction of new 

 charged groups onto the enzyme (as with iodoacetate) could favor the 

 dissociation of helices or unfolding. The reaction of inhibitors with protein 

 groups that are involved in secondary structure (e.g., inhibitors forming 

 hydrogen bonds with the protein) or tertiary structure (e.g., inhibitors react- 

 ing with sulfhydryl groups) may also labilize the enzyme. All the consti- 

 tuent atoms and bonds in a protein molecule are probably in a rather in- 

 tense electrical field originating from the positively and negatively charged 

 groups distributed in a definite pattern and the interpolypeptide or inter- 

 helical linkages maintain the structure within this field. However, if ei- 

 ther the electrical field is altered or the linkages are weakened by inhibitors, 

 the delicate equilibrium may be shifted in favor of the so-called denatured or 

 inactive forms. It may also be postulated that an inhibitor could increase 

 the stability of an enzyme if it reacts at the active site in a manner simi- 

 lar to that of the substrate since it is known that enzymes are sometimes 

 more stable in the presence of their substrates, which may by their effects 

 on the local electrical field around the active site favor structural stability. 



The question arises as to whether inactivation of an enzyme is associated 

 with general structural changes of the protein or only to local alterations 

 in the region of the active site. Probably both types occur and it is possible 

 that a local disruption of protein structure could spread over the rest 

 of the protein as a wave of instability or denaturation. In any event, it 

 is likely that some instances of inactivation are not accompanied by signif- 

 icant changes in the properties of the jDrotein as a whole. Thus enzyme inac- 

 tivation need not imply those changes usually associated with denaturation 

 of the protein, for the disturbance may remain localized. In such cases, a 

 degree of reversibility may be inherent in the inactivation and detection 

 of the inactivation may be quite diflEicult. 



The quantitative expression for the loss of enzyme activity with time 

 when both inhibition and inactivation occur simultaneously may be very 

 complex. The kinetics of inactivation have been very well presented by 

 Reiner (1959, p. 275). For a simple first-order denaturation of an enzyme: 



k 

 E ^ X 



the concentration of the inactive form is given by: 



(X) = (E,)[l - e-«] (12-49) 



