658 14. EFFECTS OF pH ON ENZYME INHIBITION 



If only HS combines with the enzyme, it is easy to show that KJ = 

 ffys'K,. Similarly, if the substrate is a dibasic acid and exists in the equi- 

 librium, S ^ HS ;=i HoS, the apparent substrate constant is given by the 

 true constant midtiplied by the appropriate pH function. Thus, if HS is 

 the active form and combines with the enzyme, K/ — f/J'Kg. 



By the active form of any component of the enzyme system, it is meant 

 that this form participates in some way in the enzymic reaction. It is the 

 form that is bound or that undergoes reaction, or both. In the case of an 

 ionizing activator, the active form is that which is bound to the enzyme 

 and is involved in the catalytic transformation of the substrate. In some 

 instances, there is no single active form, the variously ionized species par- 

 ticipating to different degrees. 



If the enzyme ionizes over the pH range studied but the substrate does 

 not, the following reactions occur: 



Ao, HE 

 E (14-27) 



A', ES -> E + P 



when E is the active form. The conservation equation is: 



(E,) = (E) + (HE) + (ES) (14-28) 



and the rate is, as usual, given by v = A'2(ES). This leads to: 



- - ^- (S) + K.[f' + ,H);g.] <"-^'" 



or, in terms of the pH function: 



^ ^ ''" (S) Tf:K. <"-'°' 



Thus here, KJ — fJK^, and when HE is the catalytically active form, KJ = 

 f/^/K^. If both the substrate and the enzyme ionize within the pH range 

 investigated, the appropriate pH functions for both must be used. If the 

 reaction occurs between E and HS, it may be shown that ii / = f^'f/JKg. 

 Similar relationships in the literature have generally been given in terms 

 of the Michaelis constant, JT,,,, instead of the true dissociation constant 

 of the substrate, K^. It might be thought that these constants would be 

 interchangeable with respect to the pH effects, but actually it is Kg to 

 which the pH functions may be applied. When iT,,, does not equal K^ 

 and is given by (^_i + k2)jki (or any more complex combination of rate 

 constants), ^,„ depends on both K^ and k.^. If both the affinity of the enzyme 

 for the substrate and the rate of breakdown of the ES complex are altered 



