VARIATION OF ENZYME INHIBITION WITH pH 693 



flections that are concave upwards near pH 8 in the curves for citrate and 

 L-hydroxy-/5-sulfopropionate are also difficult to explain in conjunction 

 with the results on the other inhibitors. It is possible, but unexpected, that 

 the mechanism of inhibition differs within this series of inhibitors. It is 

 also disturbing that the slopes of the curves are around 0.7 to 0.8 instead 

 of the theoretical 1.0. This could be attributed in part to the increase in 

 over-all negativity of the enzyme protein as the pH is increased or possibly 

 to a surface-buffering effect, the pH on or within the enzyme not changing 

 linearly with the pH of the bulk phase. Some further explanations for these 

 discrepancies and an extension of the Dixon treatment will be discussed 

 following the next section. 



Comparisons of the Relative Potencies of Different Inhibitors 



In relating chemical structure to inhibitory activity, especially in a 

 series of inhibitors acting by the same mechanism, the most informative 

 method is to determine and compare the inhibitor constants. Now, for this 

 purpose, one would be interested only in the true constants, K^, since they 

 provide information on the affinities of the enzyme for the various active 

 forms of the inhibitors. However, it is clear that by the usual methods one 

 determines in most cases an apparent inhibitor constant, K- , which may 

 be related to Ki in a complex manner involving the pH and possibly the 

 concentrations of other components of the system. The use of p^/s to 

 compare inhibitors may be very erroneous. 



It is possible that determinations of inhibitions at a single pH may give 

 apparent inhibitor constants that are related only to the p-ffa's of ionizing 

 groups, rather than to relative affinities. As an example, let us consider 

 three monobasic inhibitors whose anions are bound equally tightly to the 

 enzyme with a true ^K^ of 3. If the inhibitions on some enzyme are deter- 

 mined at a pH of 7, the apparent constants will be given by: 



Inhibitor 1 (p;^^ = 6): pi?/ = p^, - log 1.1 = 2.96 

 Inhibitor 2 (pA^„ = 7): piiC/ = pZ, - log 2 = 2.70 

 Inhibitor 3 (pZ, = 8): piiC/ = pZ, - log 11 = 1.96 



If these p^/'s were used to compare binding affinities for the inhibitors, 

 the conclusions would be completely incorrect, and if interaction energies 

 were calculated from these piiC/'s and compared with the structures of 

 the inhibitors, any deductions would be invalid. 



Massey (1953 b) pointed out in his study on fumarase inhibition that a 

 consideration of pH in such comparisons can be very important and his 

 results well illustrate these principles. Table 14-2 presents the values of 

 \}K- for the different inhibitors at four jjH's and it is evident that if rela- 



