INTERACTIONS OF SMALL MOLECULES WITH PROTEINS 313 



ing of small molecules to proteins, the following general references may be 

 consulted: Goldstein (1949). Klotz (1949, 1953), Gurd and Wilcox (1956), 

 Scatchard et al. (1954), and Edsall and Wyman (1958, p. 591). 



Competition between Ligands for Binding Sites 



It was pointed out earlier in this chapter that the binding of an inhibitor 

 to an enzyme does not occur in a vacuum but that various types of mole- 

 cules may have to be displaced from both the site and the ligand. In the 

 absence of the inhibitor, the enzyme sites are not free but may be asso- 

 ciated to a greater or lesser degree with water, physiological ions including 

 H"^, or other substances in the medium used. The over-all binding energy 

 must include the energies required for displacement of these substances 

 upon interaction of the inhibitor with the enzyme sites. These relationships 

 have been studied most carefully in the binding of small molecules to pro- 

 teins or to simpler components of the proteins, such as peptides or amino 

 acids, and it is upon studies of this kind that one must base reasonable 

 estimates of the magnitude of the effects occurring in enzyme inhibition. 



In the formation of ammonia complexes of the metal ions, such as Cu"^"^, 

 the displacement of water molecules from the ions must be considered. 

 Since the activity of the water remains relatively constant in such systems 

 it has been customary to include it in the dissociation constants, but in 

 calculating the interaction energy of ammonia with metal ions it must 

 be remembered that the dissociation constant does not represent this alone. 

 Likewise, the dissociation constant of oxyhemoglobin includes the displace- 

 ment of the water molecule that is bound to the iron atom in the absence 

 of oxygen. In the same manner, substrate and inhibitor constants include 

 such displacement reactions. The effects of pH on binding may often be 

 attributed to a competition between the H"*" and the cation whose interac- 

 tion one is measuring; this has been studied particularly in the binding of 

 Ca"^'^ and Mg"^^ to proteins (Carr and Woods, 1955). We have considered 

 the effects of the ionic atmosphere on binding but in many cases the com- 

 mon ions of the medium are bound more tightly and more specifically to 

 protein sites. K+ and Na+ ions are bound to some proteins and not to others 

 (Carr, 1956) and with greater affinity than would be expected, leading to 

 the postulate that a chelation mechanism is responsible (Saroff, 1957). 

 If this is true, it would imply a fair degree of specificity since the carboxylate 

 groups of glutamate and asparate must be close enough to the imidazole 

 groups of histidine for the chelation to occur. Serum albumin can bind sev- 

 eral simple cations and as much as forty CF ions per molecule and it is 

 likely that many enzyme j^roteins would show similar binding properties. 



The effects of different buffers and ionic strengths are perhaps to be 

 ascribed in part to such associations. We shall see that the nature of the 

 buffer may be important in enzyme inhibition (Chapter 15) and it is per- 



