rUMARASE 



275 



but evidence against this comes from the study of inhibition by the tar- 

 trates. The catalytically active form of fumarase may be represented by 

 EH, in which one of these groups is protonated, although both E and EHg 

 are capable of binding both substrates and inhibitors. 



The values of K^ for several competitive inhibitors at pH 6.35 and 23° 

 (Table 2-1) may be used as a rough and provisional means of evaluating the 

 relative energies of ])inding, bearing in mind that these are apparent K-&, 

 that the enzyme exists in three different ionized states for each of which 

 the binding is different (so that the K^s are in a sense composite), and 

 that the various inhibitors alter the pA^^'s of the enzyme groups in different 

 ways in the EI complexes. However, some reasonable conclusions may 

 be drawn from these AF values. 



Table 2-1 



Inhibitor Constants" and Relative Binding Energies 

 FOR Competitive Inhibitors of Fumarase 



Inhibitor 



Apparent K^ 

 (mM) 



Relative —AF 

 (kcal/mole) 



Adipate 



Succinate 



Glutarate 



Malonate 



D -Tartrate 



Mesaconate 



Maleate 



L-a-Hydroxy-/5-sulfopropionate * 



D-Malate 



Citrate 



<rans-Aconitate 



° Values of Ki determined at pH 6.35 and 23°. 



* The Ki for L-a-hydroxy-^-sulfopropionate was changed from 16.5 mM as given 

 in the table (Massey, 1953 b) to correspond to the value in the curve presented (16.5 

 is probably a misprint for 10.5.) 



[A) Since all monocarboxylates and the methyl ester of fumarate are 

 without inhibitory activity, it must be assumed that at least two negatively 

 charged groups are necessary for binding. However, it is evident that for 

 the more tightly bound substances other attraction forces are involved. If 

 we assume that these additional forces arise from hydrogen bonding be- 



