94 INTERMEDIARY METABOLISM AND GROWTH I 



tion with aspartate and COj yields 5-aminoimidazole carboxamide ribotide 

 (AICAR) (Levenberg and Buchanan, 1956). The amine was identified as 5-amino- 

 imidazoleribotide (AIR). By fractionating the enzymatic components, a second 

 compound, an intermediate in the conversion of FGAR to AIR, was isolated and 

 it was identified as a-N-formylglycineamidine ribotide (FGAM). 



The conversion of FGAM and of AIR to inosinic acid also takes place in the 

 presence of liver enzymes supplemented with bicarbonate, aspartate, ATP and 

 formate. ^"^CO, is fixed into these ribotides. Azaserine does not affect the conver- 

 sion of AIR to AICAR and IMP but does prevent the formation of FGAM from 

 FGAR in the enzyme system, confirming the primary locus of inhibition by this 

 substance. The enzymes of pigeon liver which convert AIR to AICAR were sepa- 

 rated into two fractions. Fraction I catalyzed the conversion of AIR, aspartate, 

 and bicarbonate, to an intermediate, 5-amino-4rimidazole (N-succinylocarboxa- 

 mide) ribotide. (Lukens and Buchanan, 1956). In experiments in which aspartate- 

 3-''*C was used, the intermediate became labelled. Enzyme fraction II converted 

 this intermediate to AICAR and fumarate. 



An active formyl-THFA derivative was also required for the formation of 

 inosinic acid from AICAR (Warren and Flaks, 1956). With serine as the donor 

 of the formyl group, potassium ions, leucovorin and pyridine nucleotides were 

 needed for IMP formation or for FGAR formation by chicken or pigeon liver 

 enzymes. The transformylation reactions were found to be reversible: 



leucovorin 



a) IMP + glycine + TPNH2 AICAR + serine + TPN* 



leucovorin 



b) IMP + GAR . FGAR + AICAR 



DPN% K^ 



An enzyme which catalyzed exchange of formate-^ "^C with IMP, stimulated by 

 leucovorin, has also been demonstrated (Flaks and Buchanan, 1954). In the 

 presence of THFA, pigeon or pig liver enzymes catalyze the formation of IMP 

 from formate-^'^C and AICAR (Greenberg et al., 1955). ATP is required to acti- 

 vate the formate. NjQ-formyl-tetrahydrofolic acid can substitute for formate and 

 ATP in IMP synthesis. 



The above formulation for the terminal steps in IMP biosynthesis is strengthened by 

 earlier studies with microorganisms. Sulfanilamide, a metabolic antagonist of para- 

 aminobenzoic acid (PABA), interferes with the synthesis of the active coenzyme form of 

 folic acid. In sulfanilamide inhibited E. coli cells, aminoimidazolecarboxamide (AICA) 

 and the riboside of this compound accumulate. y\ICA riboside is also formed by a PABA 

 requiring mutant of E. coli (Greenberg and Spilman, 1956). It is reasonable to assume 

 that the riboside arises from the ribotide, AICAR, by phosphatase action and is then 

 excreted into the medium. Small amounts of AICAR can also be isolated from E. coli grown 

 under sulfanilamide bacteriostasis (Greenberg, 1956). 



The formation of adenosine-5 '-phosphate (AMP) from inosinic acid requires aspartate, 

 and GTP (Lieberman, 1956; Carter and Cohen, 1956). E. coli enzymes catalyzing AMP 

 synthesis have been purified 40 fold. Adenylosuccinate is an intermediate in this con- 

 version. Aspartate- 1, 4- i^C is incorporated into adenylosuccinate without dilution as is 

 inosinic acid-8->''C. The requirement for aspartate in the conversion of inosinic acid to 

 AMP has also been demonstrated in bone marrow extracts (Abrams and Bentley, 1955a). 



