35. BIOSYNTHESIS OF PURINE NUCLEOTIDES 315 



When reacted with the Bratton and Marshall reagents, 5-amino-4-imi- 

 dazolecarboxylic acid ribonucleotide yields a red-purple-colored product 

 with an absorption maximum at 519 rn.fi. Like its immediate precursor, 

 5-amino-4-imidazolecarboxylic acid ribonucleotide is difficult to isolate and 

 purify. It is relatively stable in alkaline solution but in acid solution it is 

 decarboxylated. Likewise, at 100° in acid solution 5-amino-4-imidazolecar- 

 boxylic acid ribonucleotide is completely destroyed as measured by the 

 Bratton and Marshall test. 



The enzyme responsible for the carboxylation reaction, 5-aminoimi- 

 dazole ribonucleotide carboxylase, has been purified over fort yf old. The 

 equilibrium constant for Eq. (8) is in the neighborhood of 3.3 liters per 

 mole. The carboxylation reaction proceeds appreciably only in the presence 

 of high concentrations of bicarbonate or under conditions in which the 

 product of the carboxylation is removed by further reaction. 



Under physiological conditions 5-amino-4-imidazolecarboxylic acid ribo- 

 nucleotide is removed by reaction with aspartic acid and ATP to yield 5- 

 amino-4-imidazole-iV-succinocarboxamide ribonucleotide [Eq. (9)]. 51 De- 

 spite the fact that fairly acidic conditions must be used during the chro- 

 matographic isolation of this highly negatively-charged molecule, it is rel- 

 atively stable under these conditions. It has been obtained as the barium 

 salt in purified form. The succinocarboxamide ribonucleotide has an absorp- 

 tion maximum at 268 m/i. When reacted with the Bratton and Marshall re- 

 agents at room temperature the succino compound does not yield a colored 



5-Amino-4-imidazolecarboxylic acid ribonucleotide + aspartic acid + ATP ^ 



5-amino-4-imidazole-A r -succinocarboxamide ribonucleotide (9) 



-I- ADP + orthophosphate 



product as would be expected for a heterocyclic amine of this type. It is 

 now known that the failure of the succinocarboxamide ribonucleotide to 

 yield a product is due to the fact that the diazotized intermediate is unstable 

 at room temperature and breaks down before coupling with the iV-1-naph- 

 thylethylenediamine takes place. When the reaction is carried out at 0°, 

 however, a purple-colored product is formed with an absorption maximum 

 at 560 nux. The fact that the succinocarboxamide ribonucleotide is the only 

 one of the unsubstituted heterocyclic amines which does not yield a 

 colored product at room temperature has proved to be a useful tool for its 

 determination. 



Equation (9) is readily reversible. In the presence of orthophosphate and 

 ADP, S-amino^-imidazole-AT-succinocarboxamide ribonucleotide breaks 

 down to 5-aminoimidazolecarboxylic acid ribonucleotide, ATP, and aspartic 

 acid. Arsenate may replace phosphate in the reaction but ADP is still re- 

 quired in catalytic quantities to achieve maximal rate of reaction. The 



