EFFECTS OF THE DIELECTRIC CONSTANT 803 



constant of acetic acid, for example, is reduced very significantly in water- 

 methanol mixtures (Shedlovsky and Kay, 1956). By this effect the total 

 charge on the enzyme, or the charge distribution at or surrounding the 

 active center, or the fraction of the inhibitor molecules in the ionic form, 

 may be changed so that the interaction between the enzyme and the inhi- 

 bitor is modified. 



(6) The interactions of all dipolar and ionic substances in the medium with 

 the enzyme will be changed when the dielectric constant is reduced, and 

 since there is frequently a relationship between the inhibition and these 

 other substances, the inhibition may be altered secondarily. For example, 

 there may be some competition between an ionic inhibitor and similarly 

 charged ions or ionic substances in the medium for a group in the active 

 center, in which case K^ will be changed because of the greater ability 

 of these other ions to bind to the enzyme when the dielectric constant is 

 lowered. 



(c) The structure of the enzyme or of the active center may be modified, 

 particularly in higher concentrations of the organic solvent. It is well 

 known that most proteins and enzymes are denatured in organic solvents 

 and it is quite possible that minor or localized structural changes could oc- 

 cur before detectable denaturation is evident. 



(d) A reduction of the water concentration can slow certain enzyme reac- 

 tions in which water is one of the components of the reaction, and it will 

 also eventually result in a decreased hydration of both the enzyme and 

 the inhibitor. Thus the concentration of water may play a role in addition 

 to the change in the dielectric constant. 



(e) A very important factor may be the interaction of the organic solvent 

 with the enzyme or the inhibitor. Since no solvent that is miscible with 

 water can be completely unreactive or inert, there is always the possibility 

 that the solvent may interfere in the inhibition directly. Most of the or- 

 ganic solvents used can form hydrogen bonds with ai)propriate groups and 

 also can interact more or less nonspecifically by dipolar and dispersion 

 forces with the enzyme or the inhibitor. Indeed, competition between the 

 solvent and the inhibitor for the enzyme may occur. The alcohols might be 

 said to be particularly prone to affect enzyme reactions and inhibitions in 

 this manner and the examples to be discussed later will provide evidence 

 for this. As mentioned above, dioxane is perhaps less objectionable in this 

 regard than other solvents. 



(/) Finally, a question specifically related to the manner in which the 

 water-solvent mixtures are prepared may be mentioned. Enzyme reactions 

 are usually run in media containing various salts, buffers, and cofactors. 

 When additions of an organic solvent are made, should this be done to 

 the normal aqueous medium, resulting in a decrease of the concentrations 



