ANALOGS WHICH ARE ISOMERS OF SUBSTRATES 273 



not bound as tightly to the enzyme as are the substrates. When citrate is 

 3.3 mM, 50% inhibition is found with 16 xnM trans-a.comta.te; since K^^ 

 for citrate is roughly 1 mM, K, is approximately 4 mM. The inhibition is 

 competitive although the Ijv — 1/(S) plots are not ideal, perhaps because 

 of some enzyme inactivation or failure to achieve equilibrium. The inhibi- 

 tion of rat mammary gland aconitase seems to be somewhat more potent, 

 since equimolar concentrations of citrate and ^raws-aconitate lead to around 

 50% inhibition (Abraham et al., 1960). Studies on the stereospecificity and 

 deuterium transfer during reactions catalyzed by aconitase (Speyer and 

 Dickman, 1956; Englard and Colowick, 1957) point to a three-point at- 

 tachment of the tricarboxylates (and perhaps an intermediate carbonium 

 ion) to the apoenzyme and Fe++. The carboxyl groups in trans-aconitate 

 would not appear to be in such a favorable position as in m-aconitate for 

 the formation of this complex and this might explain the relatively weaker 

 binding. The effects of trans-acomtate on other enzymes have been little 

 investigated but it has been found to be a fairly potent inhibitor of fu- 

 marase, Z^ being 0.63 mM at pH 6.35 (Massey, 1953 b). The K, increases 

 with rise in the pH (Fig. 1-14-11) and, as with malonate, the formation of 

 the EI complex is exothermic at low temperatures and endothermic at 

 high temperatures. 



It is somewhat surprising that tro ns-aconitate is a reasonably effective 

 inhibitor of the respiration of intact cells, inasmuch as penetration into the 

 cells should be difficult. The following inhibitions have been observed: 

 22-36% of endogenous respiration of various tumor shces and 28% of 

 endogenous respiration of liver slices at unspecified concentration (Wein- 

 house et al, 1951), 25% of Paramecium respiration at 10 mM (Holland and 

 Humphrey, 1953), and 40% of the ion-linked respiration of barley roots 

 at 20 mM (Ordin and Jacobson, 1955). However, no inhibition of the 

 respiration of Australorbis mince at 10 mM was reported (Weinbach, 

 1953). The oxygen uptake resulting from the addition of citrate or cis- 

 aconitate to rat liver slices is strongly inhibited by 30 mM ^raws-aconitate 

 (Sherman and Corley, 1952). The most complete study of respiratory in- 

 hibition is by Saffran and Prado (1949) with rat liver and kidney slices. 

 In the latter the inhibition is 27% at 2 mM and 73% at 20 mM, which is 

 quite comparable to malonate. The inhibition by 2 mM ^rans-aconitate 

 is not altered by adding malate or fumarate, but is reversed with 5 mM 

 citrate or cis-aconitate. In other experiments the sensitivity to trans- 

 aconitate is unexplainably less. The inhibition of aconitase in liver and 

 mammary gland homogenates leads to a fairly marked depression of the 

 conversion of citrate to CO2 and of acetate to fatty acids by fmws-aconitate 

 (Abraham et al., 1960). The synthesis of mammary fatty acids is inhibited 

 45% by 7.1 mM and 75% by 21.4 mM. Accumulation of citrate accompa- 

 nies the inhibition in kidney, liver, and tumor slices (Weinhouse et al., 



