BIOSYNTHESIS OF PURINES AND PYRIMIDINES 289 



nor did it affect the incorporation of C^^ into hypoxanthine. These experi- 

 ments indicate that the carboxamide itself is not on the path of hypoxan- 

 thine synthesis, but that, instead, a derivative of it is probably involved. 



At that time Greenberg^^^^ had already made the important observation 

 that inosinic acid rather than hypoxanthine is the first purine compound 

 formed in pigeon liver systems. This made it possible to explain the results 

 obtained with the carboxamide, and both Greenberg^^ and Schulman and 

 Buchanan^^ suggested that the carboxamide ribotide might be the inter- 

 mediate in the synthesis of inosinic acid from small molecules and that 

 coupling with ribose phosphate precedes ring-closure. Further support for 

 this theory lies in the tentative demonstration by Ben-Ishai et al.^^ of the 

 formation of the carboxamide deoxyriboside from the carboxamide by a cell 

 suspension of E. coli, and in the fact that this deoxyriboside had a growth- 

 enhancing effect on a purineless mutant of E coli, which was 5 times that of 

 the free carboxamide. Furthermore, Greenberg^^ demonstrated that the 

 carboxamide riboside and not the free carboxamide is the major carbox- 

 amide component in young cultures of sulfadiazine-inhibited E. coli. Re- 

 cently Greenberg^" has also showai that in enzyme systems from pigeon liver 

 extracts or from autolysates of brewer's yeast the carboxamide riboside 

 together wdth phosphoglyceric acid forms the carboxamide ribotide. This 

 ribotide in the presence of formate can then give rise to inosinic acid. On 

 the basis of evidence from trapping experiments with formate-C^^ and non- 

 labeled carboxamide riboside, Greenberg also suggested that the riboside 

 per se is not an intermediate in the de novo synthesis of inosinic acid from 

 small molecules. The combined evidence indicates that ribose and phos- 

 phate are attached to an acyclic purine precursor at an early stage prior 

 to carboxamide formation and that it is the carboxamide ribotide which 

 lies on the direct synthetic pathway to inosinic acid (see also Chapter 24). 



Studies on the enzymic synthesis of inosinic acid have been conducted by 

 Buchanan and co-workers and by Greenberg, and have been of the greatest 

 importance. The earher-mentioned demonstration by Greenberg-^ that 

 ribose phosphate, or compounds which could give rise to ribose phosphate, 

 stimulated the synthesis of hypoxanthine from formate-C'* was the first 

 indication that inosinic acid and not hypoxanthine is the primary purine 

 product formed from small molecules. By following the incorporation of 

 formate-C^^ into hypoxanthine, inosine, and inosinic acid at different times, 

 the results represented in Fig. 5 were obtained. ^^ The figure gives the total 

 radioactivity of the compounds. Measurements of the specific activity of the 

 same compounds at early stages showed that by far the greater part of 



68 R. Ben-Ishai, E. D. Bergmann, and B. E. Volcani, Nahire 168, 1124 (1951). 

 6' G. R. Greenberg, /. Am. Chem. Soc. 74, 6.307 (1952). 

 " G. R. Greenberg, Federation Proc. 12, 211 (1953). 



