Biosynthesis of Porphyrins 65 



reversible — I know the free energies would be very much against this 

 equilibrium and would certainly be vastly in favour of the succinate — 

 but if such a reaction occurred in the cell to a small extent, it might 

 explain the findings of Dr. Shemin without assuming the 4-carbon 

 intermediate. He may have some reasons why he excluded that possi- 

 bility. 



Shemin: I am assuming the 4-carbon intermediate at least until the 

 ketoglutarate-succinate reaction is shown to be reversible. From purely 

 organic chemical lines I can't see how ketoglutarate can go to succinate 

 without intermediates, involving as it does an oxidative decarboxylation; 

 a succinyl derivative, e.g. a succinyl coenzyme complex, may very well 

 be an intermediate. I prefer this 4-carbon compound with reactive 

 groups on a terminal carbon atom. It could very well be that keto- 

 glutaric acid would explain all the findings if ketoglutaric acid were 

 derived from succinate, but so far it has not been demonstrated, as far 

 as I can gather. 



Wood: I have not seen any convincing evidence for the synthesis of 

 a-ketoglutarate from COg and succinate in animal tissues. In fact, the 

 work that has been done on the reversibility of this reaction with 

 bacteria is not so convincing that it should be accepted as a proved 

 reaction. I think that the chances of an intermediate occurring in this 

 reaction are exceptionally good. Dr. Utter in our laboratory has 

 provided evidence that there is an intermediate C3 compound in the 

 fixation of CO 2 in oxaloacetate. Until now this reaction has usually 

 been considered to be a straightforward reaction between pyruvate and 

 COj. We believe now that it is not, because pyruvate does not always 

 exchange in oxaloacetate at the same rate as COg. Dr. Utter has shown 

 that there are two enzymes involved: with one enzyme present CO2 is 

 fixed in oxaloacetate, but there is no fixation of pyruvate; if then the 

 other enzyme is added, the fixation of pyruvate and CO 2 is approximately 

 equal. 



Isn't it a misnomer to speak of acetic acid as the "source" of so many 

 carbons? Actually we probably should speak of either a-ketoglutarate 

 or succinate as the source. Have you tried labelled a-ketoglutarate to 

 see whether it will replace acetic acid? 



Shemin: I agree that we shouldn't refer to acetic acid as the source 

 of the carbon atoms, and as soon as we know the structure of the 4 

 carbon compound, we will have to call it the precursor. We are at 

 present synthesizing ketoglutaric acid and hope to see whether it gives 

 rise to the same pattern of distribution; we are, in fact, making all 

 members of the citric acid cycle. We can, however, dilute out acetate 

 incorporation by unlabelled members of the citric acid cycle, and in 

 fact can dilute out acetate to zero very nicely with the semialdehyde 

 of succinic acid. But there are objections to this type of experiment. 

 I think one must synthesize the various compounds and do the full 

 degradation. 



Wood: a-ketoglutarate does dilute it out? 



Shemin: Yes, and citrate and succinate also dilute the utilization of 

 acetate. These compounds do not damage the cell system; ^^N labelled 



ISOTOPES 6 



