INHIBITIONS OF PURIFIED ENZYMES 881 



than others. One should choose that method, or those methods, which are 

 most likely to provide accm'ate values for the constants characterizing the 

 inhibition, and then to check the determinations using different forms of 

 plotting. In this way certain abnormal types of inhibition may be uncovered 

 because not all the plots will be equally sensitive to deviations from the 

 classic kinds of inhibition. More accurate and reliable values for the in- 

 hibition constants may also be obtained. One must always bear in mind 

 that there are many possible inhibition mechanisms, some of which are not 

 easily recognizable in the common plots. It is probably a good idea before 

 interpreting the data, to list all the conceivable mechanisms by which the 

 inhibition might take place and to devise analytical procedures whereby 

 these may be distinguished. 



(6) Do not use an unnecessarily high substrate concentration. Many en- 

 zymes are inhibited by high substrate concentrations (see Chapter 4) and 

 under these conditions the responses to inhibitors will be altered. The en- 

 zyme may also be saturated at high substrate concentrations, the rate being 

 independent of the substrate concentration, and under these conditions the 

 effects of inhibitors on the binding of substrates (i.e., changes in K,.) will 

 not be so readily observed. 



(7) Compare different inhibitors by determining the true inhibitor constants. 

 In comparing the potencies of several inhibitors on a single enzyme, it is 

 not enough to determine the inhibitions at single concentrations of each 

 inhibitor, especially if one is dealing with a series of homologous compounds, 

 because it is only through the use of the true K/s that accurate results on 

 relative binding can be obtained. If, for some reason, it is not convenient 

 to determine the values ofK^, at least several inhibitor concentrations should 

 be used and the concentrations producing a specified degree of inhibition 

 should be presented. 



(8) Determine the effects of changing the pH on the inhibition. A great 

 deal of information may frequently be obtained from studies on the variation 

 of the inhibition with the pH. In relation to the suggestion immediately 

 above, it is important in comparing different inhibitors to determine the 

 true K^ values. It was shown in Chapter 14 that the inhibition expressions 

 for dissociable inhibitors involve a pH term and that the true K, is often 

 quite different from that determined by the plotting procedures. In other 

 words, the values of the apparent or experimental K^ for a series of inhibi- 

 tors may relate principally to the ionization constants of the inhibitors 

 rather than to their relative binding to the enzyme. If one is attempting 

 to correlate inhibition with chemical structure, the relative affinities of 

 the active forms of the inhibitors are desired, not merely the fractions of 

 the inhibitors in the active form at the chosen pH. Furthermore, it is 

 clear that information on the nature of the groups at the active centers of 



