VARIATION OF ENZYME INHIBITION WITH pH 707 



It can be shown in the system assumed above, where I is the active form 

 of the inhibitor, that lowering the pH below the p^,, of the inhibitor will 

 indeed reduce the inhibition, but not to as great an extent as in systems that 

 do not exhibit mutual depletion. 



The inhibition of «-chymotrypsin by diisopropylfluorophosphate in pure 

 water is reversible and competitive (Stein and Laidler, 1959). This is a 

 mutual depletion system because for inhibitions between 17% and 88%, 

 (I,)/(E/) was found to be between 0.39 and 3.54. The variation of K^ with 



the pH was determined and a maximum around a pH of 8.1 was demonstra- 

 ted. Both the uninhibited rate and the inhibition vary with pH in the same 

 fashion, which led to the conclusion that diisopropylfluorophosphate reacts 

 with the enzyme in the same way as does the substrate. The mechanism of 

 the pH eifects is interpreted in terms of a simultaneous nucleophilic attack 

 by an imidazole nitrogen and an electrophilic attack by a protonated amino 

 group. 



The pH of Inhibitor Solutions and Enzyme Preparations 



Accurate knowledge of the pH is necessary in all enzyme study and espe- 

 cially so in experiments designed to investigate the effects of varying the 

 pH. Much of the early work on enzymes and inhibition is difficult to inter- 

 pret because of the failure to control the pH adequately. In many reports, 

 unfortunately, the pH at which the experiments were run is not given, but 

 this is becoming progressively less common. Even when the pH is stated, 

 one can justifiably ask if this is reliable or whether undue faith was put in 

 some buffer system. Perhaps one will be excused for including a few 

 statements on these simple yet important problems. 



Many inhibitors are weak acids or bases and solutions of their salts 

 are often at pH's far removed from neutrality. Solutions of sodium iodo- 

 acetate, sodium malonate, and similar salts at 5 to 10 niM range in pH 

 from 8 to 9 whereas solutions of the corresponding acids are between 2.5 

 and 3.5 frequently. Even lower concentrations of the acids can decrease the 

 pH significantly; e.g., 1 mM 2,4-dinitrophenol has a pH of 3.85 and 1 niM 

 iodoacetic acid a pH of 3.26. When such solutions are added to enzyme 

 preparations, the important question is whether in the presence of the buf- 



