672 



14. EFFECTS OF pH ON ENZYME INHIBITION 



(f) E is active form of enzyme: I combines with HE. 



E +P 



HEI ;:± HE ;=± E — ES 



Vi 



VmiS) 



(S) + K, 



fe + 



(I) 



(H)(1) 



KaKi 



(I) + K, 



fne + 



^a(S) 



K.m 



(14-68) 



(14-69) 



(14-70) 



It may first be noted that in each case when the pH is in the range 

 where the enzyme is predominantly in the form combining with the inhi- 

 bitor, the inhibition becomes classically competitive or noncompetitive. 

 In the first fom- cases, this is the pH range where the enzyme is in the ac- 

 tive catalytic form; in the latter two cases it is when the enzyme is in the 

 inactive form. Secondly, it is seen that when the inhibitor combines with 

 the enzyme noncompetitively (last four cases), the inhibition depends on 

 (S), contrary to what is generally thought to be true for noncompetitive 

 inhibition. Now, in these cases, the inhibition depends on (S) only in a 

 certain range of pH: in (c) and (f) when the pH is significantly higher than 

 the pZ, and in {d) and (e) when the pH is significantly lower than the \)K^. 

 As the pH is changed from one side of the y>K„ to the other, the character 

 of the inhibition will thus alter. Thirdly, it may be observed that it might 

 have been predicted that K- = fJK^ or K- = f^/K,, depending on the 

 form of the enzyme with which the inhibitor reacts, but this is only true 

 when the substrate concentration is significantly lower than K^. The im- 

 portance of this will be discussed when the Dixon treatment of inhibition 

 is presented. 



This behavior must now be explained on a physical basis. In the first 

 place, why does the inhibition in such cases depend on the pH at all? One 

 might suppose that since both v and v^ are modified by pH due to shifts 

 in the equilibrium, E ^ HE, that the inhil)ition would remain constant. 

 Let us examine the situation depicted in (a) above. From Eq. 14-55, it 

 is seen that when the pH is sufficiently below the pX,,, so that the term 

 in brackets in the denominator becomes essentially 1 -f [(S)/iiL,], the in- 

 hibition is independent of pH change; in a higher pH range, the hydrogen 

 ion may become a more important factor than the sul)strate. Now, when 

 the pH is low, most of the enzyme is in the HE form and the addition of 

 an inhibitor will reduce (HE) in a simple mass-action manner; however, 

 at higher pH's, when the enzyme is ])redominantly in the E form, the ad- 

 dition of inhibitor will reduce (HE) with the result that E -> HE. The con- 

 centration of HE is in a way buffered by the reserve of E. As long as there 



