ROBERT A. ALBERTY 



values. Proteins are polybasic acids and because of the large 

 number of ionizable groups the effect of /?H on the kinetics may 

 be extremely complicated. In the neutral pH range proteins 

 consist of a very large number of isomeric forms. For example, 

 if A of a total of m acidic groups are ionized there are m \/{m-h) ! h ! 

 different ions corresponding to the number of different ways in 

 which it is possible to choose the h protons from m groups. 

 However, it is to be expected that the ionizable groups in the 

 neighborhood of the active site will exert a preponderant effect. 

 If the gain or loss of a proton by a group in the catalytic site 

 causes a total loss in enzymatic activity, the primary effect of 

 changing the pYi may be represented quite simply. The idea 

 that the bell-shaped plots of activity versus /?H are a consequence 

 of the changing degree of ionization of the enzyme was originally 

 suggested by Michaelis. In order to account for a maximum in 

 such a plot it is necessary to postulate that there are at least two 

 ionizable groups in the enzymatic site. If the enzyme-substrate 

 complex breaks down to yield product only when there is a proton 

 on one group and not on the other, a symmetrical maximum 

 initial velocity versus /?H plot will be obtained and the ionization 

 constants of the two groups may be obtained from the experi- 

 mental data, 



Fumarase has proved to be a very suitable enzyme for a 

 detailed study of the effect of /?H. The reaction catalyzed is 



F + H2O ;=± M (13) 



where the symbols F, fumarate, and M, /-malate, include the 

 various ionized forms of the substrates. The effect of /?H on the 

 reaction rate indicates that the catalytic activity is dependent 

 upon two ionizable groups. The experimental results may be 

 accounted for in terms of the following mechanism, which is the 

 simplest mechanism which will represent the effect of />H on the 

 maximum initial velocities and Michaelis constants for both the 

 forward and reverse reactions (7). 



574 



