FUMARASE 277 



tively weakly (0.3-0.6 kcal/mole). In fact, the low interaction energies of the 

 terminal COO" groups (0.87 kcal/mole for each group, which may be com- 

 pared with the 3.3-3.6 kcal/mole binding per COO" group of malonate or 

 succinate on succinate dehydrogenase) might indicate that the distance 

 between them and the enzyme cationic groups is relatively great (perhaps 

 12-15 A), or could even point to a type of interaction other than ion-ion 

 attraction. 



(D) L-a-Hydroxy-/?-sulfopropionate is bound at least 0.54 kcal/mole less 

 tightly than L-malate, much of the affinity of this analog resulting from 

 the OH group interaction. This would indicate that the sulfonate group 

 is not a very good substitute for a COO" group in this case. It would be 

 interesting, in this connection, to have inhibition data on L-/5-sulfopropio- 

 nate. 



(E) When one turns to the effects of pH on the binding of these inhi- 

 bitors, it is evident that the situation is more complex than assumed from 

 the data at a single pH (see Fig. 1-14-11). Several inhibitors exhibit a pro- 

 gressive decline in binding with increase in the pH, but in the case of fu- 

 marate the pZ,„ rises between pH 7 and 8, and the pZ,„-pH curve for l- 

 malate shows several changes of slope. The inhibitor D-malate also shows 

 an increase in binding between pH 7 and 8. Massey (1953 b) suggested that 

 binding to different sites might be involved. However, since deviant be- 

 havior is noted with substances containing a double bond or OH group, 

 it is possible that the affects of pH on the polarization and hydrogen bonding 

 interactions may be involved. Succinate exhibits a linear decrease in jiK^ 

 with pH whereas fumarate behaves quite differently. Mesaconate and ma- 

 leate have succinate-type pH dependences and it is possible that the pola- 

 rization interaction is sterically prevented in these substances, as postulated 

 above. Apparently some change in the active center ionization occurs 

 between pH 7 and 8 which alters these interactions, and we shall return to 

 this problem later when more recent inhibition data have been presented. 

 (If the crude approach to these problems in the preceding paragraphs 

 serves either to irritate or activate others to further theoretical or experi- 

 mental study, a purpose will have been accomplished.) 



One would expect the most potent competitive inhibitors of fumarase 

 to have either a polarizable group (such as — C=C — ) or a group capable 

 of forming hydrogen bonds (such as OH). Substitution of groups at the 

 double bond of fumarate seems to reduce the binding, and acetylene-di- 

 carboxylate has not been studied. Thus one is left with the tartrates as 

 possibly interesting inhibitors, and they were investigated by Wigler and 

 Alberty (1960) in an excellent study designed to establish the more intimate 

 nature of the catalysis. The variation of the inhibitions with pH allowed the 

 determination of the p^^'s of the enzyme groups, the changes in these pro- 

 duced by complex ing with the inhibitors, and the dissociation constants 



