710 14. EFFECTS OF pH ON ENZYME INHIBITION 



It was assumed that buffer ions could bind to the enzyme competitively 

 and noncompetitively with regard to the substrate, the former being re- 

 presented by EB and the latter by BE. The inhibition is competitive and 

 hence BEI may be formed but not EBI. The situation is complex, the data 

 are difficult to interpret, and experimentally there is no way to avoid such 

 effects (unless it is possible to find another buffer with the proper pK^ 

 and which does not interact appreciably with the enzyme). 



The complications sometimes introduced by the buffer make it reason- 

 able to reduce the buffer concentration as much as possible consonant with 

 the system used. Some enzymic reactions do not alter the pH as they pro- 

 ceed and some can be measured before any pH changes might occur; in 

 such cases, high concentrations of buffer are superfluous. If each of the 

 solutions that must be mixed to give the final enzyme preparation can be 

 adjusted to a single desired pH, the presence of a buffer is not so impor- 

 tant. In many cases, 0.01 to 1.0 M buffers are used without reason, where- 

 as the concentrations could be reduced to minimize any buffer effects.* 



Suggestions for pH Studies of Inhibition 



Certain practical points may be summarized from the discussions covered 

 so far in this chapter. These may be useful in planning investigations of 

 enzymes and their inhibition as related to pH and thus are itemized here. 



A. Many reports deal with the characterization of purified enzymes. In 

 most of these studies, the rate is determined at several pH's and the V^opt 

 given. This is a useful figure but it is not as informative as the p^^'s of 

 ionizing groups at the active center, since from these some knowledge of 

 the nature of the groups participating in the catalysis can often be obtained. 

 Similarly, when certain types of inhibitors are used, it is not difficult to 

 determine K/ at a few pH's and, by suitable plotting, to arrive at a better 

 understanding of the chemical make-up of the active center. 



B. Inhibition studies of an enzyme should be made at a pH approximating 

 that in the cells from which the enzyme was obtained, if this pH is known 

 or can be estimated, whatever the 'pH-opt of ^^e enzyme. Only data obtained 

 at a physiological pH can be applied to inhibitions in the living cells. 



C. The reversibility of pH effects should be checked in order to recognize 

 and eliminate denaturation and other irreversible inactivation processes; 

 unless this is done, the data cannot be correctly interpreted by the kinetic 

 and plotting procedures discussed above. 



* It is interesting that in the Methods in Enzymology (Colowick and Kaplan, 1955) 

 the buffer concentrations used in the standard enzyme assays average 0.057 M and 

 in 18% the buffer concentration is 0.1 M or over. 



