654 14. EFFECTS OF pH ON ENZYME INHIBITION 



from theories based only on active center groups may well arise in these 

 more indirect influences. The attempts to estimate the effects of these 

 outlying groups will be considered later. 



The division of the mechanisms with regard to the steps in the catalytic 

 sequence (as in category I) can be illustrated in the reaction: 



E+S;^ES^EP-^E+P (14-19) 



where only the forward formation of product is considered. The rate con- 

 stants, kj^ and ^_i, may be sensitive to the pH and hence Kg = k_ilki 

 may vary with the pH. This means that the affinity of the enzyme for the 

 substrate is pH-dependent because of changes on the groups responsible 

 for the binding (lA). The rate constant, A'g, for the dissociation into the 

 free enzyme and the product may also be pH-dependent because of the 

 changing electrical fields arising from ionizable groups at or near the active 

 center (IB). The actual molecular rearrangements occurring in the trans- 

 formation of the substrate to the i^roduct may also be pH-dependent, most 

 commonly because of the participation of dissociable hydrogen ions in the 

 reaction (IC). In those cases in which the reverse reaction is significant, 

 consideration must also be given to the possible variation of the equili- 

 brium constant between S and P with the pH. Reactions that are more 

 complex, involving additional components, will, of course, present other 

 possibilities for pH effects. 



The modifications in enzyme structure brought about by variation in the 

 pH (IV) are little understood and are not susceptible to quantitative analy- 

 sis. The structural changes may l)e reversible or irreversilile. In the latter 

 case, it is at least possible to detect such changes and take them into ac- 

 count when subjecting the data to kinetic theory. It is important in any 

 study of pH effects to eliminate these irreversible modifications in the en- 

 zyme (usually termed denaturation). Every enzyme will become unstable 

 outside of a particular and characteristic pH range. It is necessary to ex- 

 amine, under the conditions of the procedure, the reversibility of the changes 

 induced by the pH and thus establish that one is working within a pH 

 range susceptible to kinetic treatment. The return of the enzyme prepa- 

 ration to the initial pH to demonstrate any loss of activity is the most 

 common method. Sometimes the instability of an enzyme may be detected 

 by a progressive decline in the rate at abnormal pH's, and thus it is advisable 

 to determine the activities or inhibitions over different time intervals when 

 a system is first being studied. Eventually it may be necessary to limit the 

 measurements to a short initial period before an appreciable irreversible 

 change has occurred. The evidence for reversible structural changes, par- 

 ticularly if they are localized, is difficult to obtain but it would appear 



