EFFECTS ON TRICARBOXYLIC ACID CYCLE 83 



5 mM malate. However, when the malate concentration is between 0.1 and 

 1 mM, the inhibition is close to 50% (Montgomery and Webb, 1956 b). 



(6) Inhibition of a-ketoglutarate oxidation. The possible inhibition of a-ke- 

 toglutarate oxidase by malonate is important not only because of the bearing 

 it has on the effects of malonate on the operation of the cycle, but also 

 because malonate has been frequently used to block succinate oxidation 

 in order to study in particulate systems the oxidation of a-ketoglutarate 

 uncomplicated by further oxidations. This technique was first proposed by 

 Ochoa (1944), who showed that high concentrations (25-50 mM) of malon- 

 ate would allow a-ketoglutarate to be oxidized to succinate in enzyme pre- 

 parations from cat heart. In four experiments with 50 mM malonate, 0.86 

 mole of succinate was formed for every mole of a-ketoglutarate utilized, 

 indicating that even here an appreciable fraction of the succinate formed 

 is further oxidized. No data were given as to whether these concentrations 

 of malonate inhibit the utilization of a-ketoglutarate. Slater and Holton 

 (1954) used 10 mM malonate to study the oxidation of a-ketoglutarate 

 in heart mitochondria, and it was shown that this concentration does not re- 

 duce the utilization of a-ketoglutarate, although 20-40 mM does inhibit it. 

 Malonate was also used to investigate the formation of a-ketoglutarate from 

 citrate in Micrococcus sodonensis (Perry and Evans, 1960), but the rationale 

 for this is obscure since malonate by inhibition of succinate oxidation would 

 not depress the disappearance of a-ketoglutarate. However, at the concen- 

 tration of malonate used (75 mM), it is quite possible that the a-ketoglu- 

 tarate oxidase was inhibited. 



The oxygen uptake with a-ketoglutarate as the substrate in particulate 

 preparations from several sources has been shown to be reduced by malonate 

 as expected if the succinate formed is partially protected from oxidation 

 (see Table 1-14). Malonate concentrations around 10 mM inhibit 40-60% 

 in most cases. No definite information on the possible inhibition of the 

 a-ketoglutarate oxidase can be obtained from such studies. 



If the oxidation of a-ketoglutarate stops at fumarate, the system is a 

 two-step linear chain (neglecting the other reactions involved in the forma- 

 tion of succinate). An atom of oxygen is taken up in each step: 



1/2 Oa 1/2 O. 



a-Ketoglutarate -> succinate ->■ fumarate 

 (1) (2) 



SO that a complete and specific inhibition of reaction (2) would result in a 

 maximal inhibition of 50% with respect to the oxygen uptake. However, in 

 case the first reaction is much faster than the second, malonate would not 

 inhibit the initial rate of the reaction, even though it inhibited the final total 

 oxygen uptake. Therefore, the inhibition may theoretically vary from to 



