INHIBITION OF SUCCINATE DEHYDROGENASE 39 



this strong inhibition is still not clear but Hellerman and his group have 

 advanced some interesting speculations. Oxalacetate in aqueous solution 

 may exist around neutrality in three forms in equilibrium. It was suggested 

 that the enolate form may be the potent inhibitor, especially the trans- 



'CX)C coo" ^ "OOC^ H 



^c=c'^ -* »^ 'ooc-c-CH,— coo" -* ^ ,c=c^ 



-Q^ ^n 'o^ ^coo" 



cJs -Enolate Keto tautomer trans -Enolate 



tautomer tautomer 



enolate tautomer where the enolate group and the carboxylate group are 

 on the same side. Evidence for this was provided by showing that the 

 oxalacetate monoethyl ester is inhibitory (about as potent as malonate) 



EtOCO^ H 



^C = C^ 



~o^ ^coo' 



^raws- Enolate 

 / tautomer of 



monoethyl ester 



(Table 1-8). The diethyl ester in inactive. Actually there is a difference in 

 binding energy of about 2.1 kcal/mole between oxalacetate and either 

 malonate or the monoethyl ester. This extra energy might be due to the 

 doubly negative charge on one end of the molecule. It could well be that 

 both the cis- and ^ra ws-enolate tautomers are bound, the charge distri- 

 butions being almost equivalent if one takes the center of negative charge 

 as lying between the carboxylate and enolate groups. It is unfortunate that 

 the per cent of the oxalacetate in the enolate forms at pH 7.6 and 30° in 

 aqueous solution is not known. Another possibility is that the keto tautomer 

 combines with the enzyme, the extra binding energy arising from an inter- 

 action between the keto group and the enzyme; a hydrogen bond could ac- 

 count for the 2.8 kcal/mole difference between the binding energies of ox- 

 alacetate and succinate. One difficulty in assuming the enolate form as the 

 active inhibitor is the fact that maleate inhibits very poorly and fumarate 

 not a great deal better. The difference in binding energy between oxalace- 

 tate and fumarate for the pig heart succinate dehydrogenase is close to 

 4.8 kcal/mole, and it would be difficult to account for this large difference 

 simply on the basis of additional ionic interactions. A final possibility must 

 be entertained, although no evidence for it exists, namely, the complexing 

 of the enolate group with one of the nonheme iron atoms near the cationic 

 groups of the enzyme surface, since this could provide the extra energy 

 for the binding of oxalacetate. It should be noted that although oxalacetate 

 strongly inhibits succinate oxidation in rapidly respiring liver mitochondria, 

 both coupled and uncoupled, it stimulates when the mitochondria are in a 



