312 6. INTERACTIONS OF INHIBITORS WITH ENZYMES 



(c) The stability of the inhibitors in the preparation used should often 

 be checked so that differences in rates of concentration decrease over the 

 period of measurement will not be interpreted as due to different degrees 

 of inherent inhibitory potency. 



{(l) The possibility of different rates of penetration into cellular or tissue 

 preparations should be considered before attributing differences in inhi- 

 bition to actions upon an enzyme system. 



(e) The attempt should be made to determine if different inhibition 

 mechanisms may be involved with different members of a series. The estab- 

 lishing of competitive inhibition, for example, with one member does 

 not ensure that this mechanism holds for all the members. 



(/) When groups are added, eliminated, or modified in an inhibitor, great 

 care should be taken not to attribute the differences observed solely to 

 the group itself, but the effects of this group change on the rest of the 

 molecule should be considered. 



INTERACTIONS OF SMALL MOLECULES WITH PROTEINS 



A great deal of very fine work in recent years has been done on the 

 binding of molecules of all types to proteins, but the results and general 

 principles that have emerged have been little applied to the problems of 

 enzyme inhibition. It has often been felt that the types of binding are 

 distinct; that measurements of over-all binding to proteins (such as would 

 result from equilibrium dialysis studies) refer to a nonspecific interaction, 

 while inhibition studies relate to specific interactions. It is true that com- 

 petitive inhibition on substrate sites by substances closely related struc- 

 turally to the substrates is probably quite specific; on the other hand, many 

 inhibitions are certainly not specific for any active site. Likewise, over-all 

 protein binding can be quite specific or relatively nonspecific. The inhibi- 

 tion of enzymes by heavy metal ions is generally not s])ecific in the sense 

 that these metals combine only with active sites; they bind at many sites 

 on the protein and incidentally at or near the active sites — a study of 

 the binding by physical or chemical methods can be very informative and 

 applicable in many respects to enzyme inhibition. The basic principles 

 involved and the types of forces operative are certainly the same in both 

 cases. In fact, the binding energies of inhibitors and small molecules gen- 

 erally cover very much the same range. Unfortunately, the subject of 

 the general binding of substances to proteins is so large that it could not 

 be adequately treated in the present work. It may be worthwhile, however, 

 to mention several of the concepts that have been derived from such bind- 

 ing studies and which bring some insight into the mechanisms and forces 

 involved in enzyme inhibition. For more detailed information on the bind- 



