316 6. INTERACTIONS OF INHIBITORS WITH ENZYMES 



positive charge dominates and where binding of a negative ion can occur. 

 Thus the net charge on an enzyme should not be considered to be as im- 

 portant as the regional charge or electrical field at the active sites, with 

 respect to the interaction of ionic inhibitors. 



One method of determining the nature of the protein groups involved 

 in the binding of a particular ligand is to react certain of the protein groups 

 and note the effect this has on the binding. A decreased binding following 

 acetylation of free amino groups on the protein might be considered as 

 indicating the participation of these amino groups at the binding sites, but, 

 as has been pointed out by Levedahl and Bernstein (1954), such acety- 

 lation also results in a reduction of positively charged groups and may 

 change the net charge of the protein from positive to negative. This could 

 have a nonspecific effect of repelling anions and be the explanation for the 

 decrease in binding. Modification of charged groups on proteins must then 

 be considered from this point of view also in attempts to determine the 

 groups with which inhibitors react. 



Effect of Temperature on Binding 



The binding of various substances to serum albumin is not markedly af- 

 fected by temperature changes (Klotz, 1949; 1953) and in some cases it 

 has been difficult to determine accurately the change in dissociation con- 

 stant over a range of 20^ to 30^. Since d In KjdT = JH!RT\ it would 

 appear that the enthalpy of binding is low in these cases and thus that the 

 affinity for the protein is mainly dependent on entropy changes. It is true 

 that the substances studied were mainly anions so that entropy changes 

 due to water displacement would be anticipated, but it is likely that 

 binding of nonionic substances often involves appreciable changes in the 

 water structure and that temperature coefficients will be relatively low 

 for these. In conformity with these results, we shall find that enzyme in- 

 hibition is frequently little dependent on temperature and probably for 

 the same reasons. Of course there are types of inhibition which represent 

 more than mere binding to the enzyme and for which high temperature 

 coefficients have been found. 



Specificity in Simple Protein Binding 



That protein complexes with small molecules are not instances of simple 

 surface adsorption or nonspecific binding is indicated in several ways. The 

 number of binding sites is often small relative to the number of molecules 

 that could cover the protein surface and the data support the hypothesis 

 that the affinity for these sites (the intrinsic dissociation constant) is 

 constant for a substance, except where interactions between sites occur. 



