Biosynthesis of the purines. III. Reactions of formate and inosinic acid and an effect of the citrovorum factor.

Previous experiments have shown that when hypoxanthine is synthesized de novo from formate, glycine, and CO2 in extracts or homogenates of pigeon liver these substrates are utilized for purine synthesis in the molecular ratio of 2: 1: 1 (1). Formate is the precursor of carbon atoms 2 and 8 of the purine base; glycine, of atoms 4, 5, and 7; and CO2 of atom 6 (2, 3). The present problem originated with the observation that when non-radioactive inosinic acid was added to the incubation mixture radioactive formate was sometimes incorporated into the purine base of inosinic acid in far greater quantities than would be expected from the measurement of de novo synthesis of purine when radioactive glycine was used as the tracer. In vessels which had been incubated with radioactive formate the specific activity of carbon atom 2 of inosinic acid was sometimes considerably greater than that of carbon atom 8. These facts led to the conclusion that in certain cases formate was involved in reactions other than those of de nova synthesis of purine, and suggested that inosinic acid-2-Cl4 may be formed by the enzymatic equilibration of radioactive formate and normal inosinic acid, 4-amino-5-imidazolecarboxamide ribotide being an intermediate of these reactions. The involvement of this latter compound in purine synthetic reactions has also been indicated by previous experiments in this laboratory with radioactive 4-amino-5-imidazolecarboxamide (4, 5) and by the studies of Greenberg (6) on the r&e of inosinic acid in hypoxanthine formation. Since it has been anticipated that folic acid derivatives participate as cofactors in the formylation reactions of purine synthesis (7), folic acid and its metabolic derivative, the citrovorum factor, were tested for their effect on the incorporation of radioactive formate in the above system.