738 14. EFFECTS OF pH ON ENZYME INHIBITION 



to those observed on the isolated enzymes. The calculation of ^^ in such 

 cases may sometimes be done by the usual graphical procedures. However, 

 the true K^ cannot be determined if the inhibitor is a weak acid or base, 

 unless the concentrations of inhibitor within the cell can be determined 

 directly. It is clear that it is under only special conditions that (I)^ = {l,)^, 

 namely when pH^ = pH^ for completely buffered cells or when the pH^ 

 is very low for unbuffered cells (see Fig. 14-16 and 14-17). In the latter 

 case, the pH^ will have altered sufficiently so that any K^ determined will 

 relate only to the inhibition at this abnormal pH. Furthermore, under the 

 best conditions, the inhibitor constant that is obtained will always be the 

 apparent one, namely K- , defined earlier in this chapter. If cellular inhi- 

 bition data are plotted by one of the usual methods that have been de- 

 veloped for isolated enzymes, the curves may superficially resemble those 

 obtained in vitro, but the constants calculated from the slopes or intercepts 

 will have no predictable relationship to the true constants of the inhibition. 

 It follows from this that a comparison of inhibitor potencies using con- 

 stants determined in this way must be unreliable. Such apparent con- 

 stants may be useful in characterizing inhibitions and providing quanti- 

 tative descriptions of the behavior, but they should not be construed as 

 expressing the affinity of the affected enzyme for the inhibitor. 



In certain cases, the calculation of K^ from data on cellular inhibition, 

 without too great an error, is possible. An inhibition that is noncompetitive 

 and potent (so that the pH^ will not change appreciably) will depend on 

 the total external concentration of inhibitor in the following manner: 



^^'^^ (14-175) 



a^ ^K ^^^' 



^^'^'^^'-m. 



1 + («^' 



K. 



Thus the experimentally determined K- will be related to the true iT, in a 

 simple manner. The relation for unbuffered cells is not simple and in cases 

 of competitive inhibition, the ionization and penetration of the substrate 

 must also be considered. 



Depletion of Inhibitor from the Extracellular Medium 



Cellular inhibition by inhibitors that are weak acids or bases and the 

 pH dependence of this inhibition are modified when the total concentration 

 of the inhibitor in the external medium is reduced as a result of the pene- 

 tration into the cells or to the binding of the inhibitor there. Cowles and 

 Klotz (1948) considered this factor in nitrophenol bacteriostasis and con- 

 cluded that it would alter the curves resulting from plotting log (I^)^ 

 against pH^, accounting for the observed slopes of less than one. Since 

 the depletion of external inhibitor could easily occur under many experi- 



