ROBERT A. ALBERTY 



the mechanism the samie values of pK^^. ^^^ P^hE were actually 

 obtained from kinetic data for the forward and reverse reactions. 



The ionization constants for the complexes of fumarase with 

 fumarate and /-malate are significantly different, and an interest- 

 ing consequence of the shift in ionization constant induced by 

 substrate is that the enzyme-substrate complex will contain a 

 different number of protons on the average than the free sub- 

 strate and enzyme molecules at the pH. of the reaction medium. 

 In other words, hydrogen ions will be produced or consumed 

 in the reaction of substrate with the enzyme. 



When the reactions of enzyme and substrate and hydrogen 

 ions are interdependent in this way, they are "linked" functions, 

 according to the terminology of Wyman (16). If the binding of 

 substrate at the active site affects the ionization of groups of the 

 enzyme, the ionization of these groups must conversely affect 

 the affinity of the enzyme for the substrate. These reciprocal 

 effects in the oxygen-binding-/?H relation for hemoglobin are 

 well known It is interesting that the effect of /?H on the fuma- 

 rase reaction is quite similar to the effect on the oxygen binding 

 by hemoglobin and that two imidazole groups appear to be in- 

 volved in both cases. 



The question arises as to why it is that two groups are 

 essential for the catalytic activity and that the intermediate 

 ionized form is the active one. A very attractive explanation is 

 that these two groups per enzymatic site are essential because 

 they are actually involved in the transfer of protons in the enzymatic 

 reaction. In EHF one ionizable group can donate a proton at one 

 alpha carbon of fumarate and the other can receive a proton 

 from a water molecule to make available a hydroxyl group at the 

 other alpha carbon of fumarate. This mechanism offers an 

 explanation of the fact that the complexes containing two 

 protons or no protons are catalytically inactive. In strongly 

 acidic solutions the catalytic site spends a large fraction of the 

 time with a proton on the group which functions as a proton 

 acceptor in the catalytic process, and during this time the re- 

 action cannot occur. In strongly alkaline solutions, the catalytic 



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