522 11. LOCALIZATION OF THE SITE OF INHIBITION 



the more certain is one that the effects are related to this particular 

 inhibition. 



Demonstration of Inhibition on Isolated Enzymes 



Ideally in the localization of action of a new inhibitor, the analysis 

 proceeds from complex metabolic systems to the sensitive enzyme. That is, 

 one first shows that the inhibitor acts on some major process in the cell, 

 then on some particular pathway, then narrows the site to a region of this 

 pathway, and finally shows the necessary inhibition on a single enzyme. 

 In such a procedure it is essential that the nature of the inhibition on the 

 single enzyme or reaction be established if one is to be completely confident 

 of the localization. It is admitted that this is not always immediately 

 possible because, as has been discussed, the site may be a previously un- 

 known component or an enzyme that has never been isolated in an active 

 state. In such cases the final localization will be achieved only with the 

 characterization of this site. 



When the approach begins w^ith and restricts itself to a study of isolated 

 enzymes, only very tenuous conclusions can be drawn. Even if all the known 

 enzymes were tested and the spectrum of inhibition established on these, 

 there are reasons why it would be impossible to deduce with validity a site 

 or sites of action in the cell. In the first place, there may be again an un- 

 known enzyme that is more sensitive to the inhibitor than any of those 

 tested. In the second place, isolated enzymes and the same enzymes within 

 the cell do not necessarily behave identically (page 436). There must be 

 evidence from work on living systems that the metabolism within which 

 the particular enzyme operates is disturbed by the inhibitor. It might be 

 said that the demonstration of inhibition of single enzymes is a necessary 

 but not sufficient proof of localization. 



A very common problem arises when a substance is found to inhibit an 

 enzyme that could well be involved in the action of the substance, but only 

 at a concentration that is appreciably higher than that required to produce 

 the action in a living system. Since few inhibitors are accumulated within 

 cells, the explanation proposed is usually that the enzyme is more sen- 

 sitive in its intracellular state and environment, which is more satisfying 

 than to admit the more likely alternative that this enzyme is actually not 

 involved primarily in the inhibition. Of course, the enzyme may be inhibited 

 more readily in the cell but at least some indirect evidence must be presented 

 that this is so if the hypothesis is to be taken seriously. Nevertheless there 

 are many instances where unwarranted deductions as to the site of action 

 have been taken seriously with the danger that they become eventually 

 incorporated into the structure of demonstrable facts and proved mecha- 

 nisms. 



