EFFECTS OF MALONATE ON PORPHYRIN SYNTHESIS 161 



in the porphyrin from acetate-C^^Hg should be altered by malonate. 

 Labeling in the carbon atoms of the A and B pyrrole rings of protoporphyrin 

 occurs after incubation of duck erythrocytes with acetate-C^^Hg and glycine. 



CH, 9 



II 

 6 H3C CH 8 



' I I 



' fi^N \ 



I 



H 



Acetate-Ci^Hg will lead directly to -OOC-C^^HaCHa-CO-CoA and if the 

 cycle is blocked completely by malonate, carbons 6 and 9 only will be 

 labeled, except for some labeling of carbons 4 and 8 due to the reversible 

 reaction succinyl-CoA :^ succinate (as long as ATP is available). Carbons 2, 

 3, and 5 should not be labeled. This is essentially seen in the tabulation. 

 The cycle, of course, is not blocked completely so that some labeling in 

 carbon 5 occurs. The over all inhibition is due to a depression of the entry 

 of acetate into the cycle. These experiments not only show the variable 

 effects of malonate on a pathway associated with the cycle but well illus- 

 trate the use of an inhibitor to elucidate a metabolic pathway. 



The analysis of the action of malonate on porphyrin synthesis was ex- 

 tended by Granick (1958) in his work with chicken erythrocytes. The for- 

 mation of protoporphyrin is innibited 90% by 10 mM malonate when only 

 glycine is present, 85% when succinate is added, and 80% when a-keto- 

 glutarate is added. The effects of different concentrations of malonate are 

 shown in Fig. 1-17. Malonate could decrease the incorporation of succinate 

 into porphyrin by blocking the cycle and reducing the ATP level, and thus 

 inhibit both pathways of succinate-CoA formation from succinate. However, 

 the quite strong inhibition of protoporphyrin formation from glycine 

 -f a-ketoglutarate is surprising. Inhibition of the step a-ketoglutarate — * 

 succinyl-CoA is not likely as the only explanation, because 1 raM malonate 

 inhibits protoporphyrin synthesis 32% and there is no reason for thinking 

 that this low concentration would inhibit a-ketoglutarate oxidase. The for- 



