Inosinic acid from formate

Similar results by Schulman and Buchanan indicated that inosinic acid was an intermediate in the synthesis of hypoxanthine from labeled glycine. Although the reactions of inosinic acid and formate are more involved than originally thought (infra vide), there is agreement that Buchanan, J. M., and Schulman, M. P., J. Biol. Chem. 202, 241 (1953). 

The synthesis of inosinic acid (123) from AIR (106) using soluble avian liver enzymes has been shown to proceed in several steps. The first step involves the formation of C-AIR (107) by carboxylation of the aminoimid-azole (106) (Scheme 15) (57JA1511). 

Fig. 14-39 Formation of urate/uric acid from inosine and guanine.

When RNA of tobacco mosaic virus is incubated with nitrous acid, changes occur in the base composition because nitrous acid deaminates cytosine and adenine. (It also deaminates guanine, but not in the intact virus.) Cytosine is converted to uracil and adenine is converted to hypoxanthine (adenylic acid inosinic acid). Consequently, the six amino groups of adenine and cytosine are replaced by keto groups. Since the complementariness of the newly synthesized RNA chain is dictated by the formation of hydrogen bonds with the bases in the template, the newly synthesized RNA is different from the wild RNA and thus a base mutation has been introduced experimentally. 

Amino-4-imidazole carboxamide ribotide, a precursor only two steps removed (formylation and cycli-zation) from inosinic acid, can be synthesized by the direct condensation of the imidazole with 5-phosphori-bosyl pyrophosphate. The enzyme catalyzing this reaction was purified from an acetone powder of beef liver. The same enzyme (AMP pyrophosphorylase) catalyzes the condensation of adenine, guanine, and hypoxan-thine. Nucleoside phosphorylase is an enzyme that catalyzes the formation of a ribose nucleoside from a purine base and ribose-1-phosphate. Guanine, adenine, xanthine, hypoxanthine, 2,6-diaminopurine, and aminoimidazole carboxamide are known to be converted to their respective nucleosides by such a mechanism. In the presence of a specific kinase and ATP, the nucleoside is then phosphorylated to the corresponding nucleotide. 

Tissues were homogenized in phosphate buffer and sonicated, and the supernatant was used for electrophoresis. Samples were run in phosphate buffer, pH 8.5, on cellulose acetate paper for 2 hours at 4 and at 0.5 mA/cm. PRPP synthetase was located on the paper by a radiochemical assay formation of PRPP from ribose-5-phosphate and ATP was coupled to inosinic acid (IMP) synthesis by the addition to the reaction mixture of labelled hypoxanthine and partially purified hypoxan-thine-guanine phosphoribosyltransferase (HGPRT). 

When a comparison was made of inosinic acid synthesis from glycine-1-C and HC OOH in the presence of added nonlabeled inosinic acid, it was observed that radioactive formate was incorporated into the acid far in excess of that anticipated from the amount of glycine utilized.i " For example, instead of a 2 1 molecular utilization of the formate glycine... 

In order to observe regularly the enzymatic exchange reaction, it was necessary to add inosinic acid to the extract and limit de novo synthesis by omitting bicarbonate from the system. When the incubation was carried out in the absence of both bicarbonate and added inosinic acid, labeled glycine and formate were converted into inosinic acid in the ratio expected from de novo synthetic reactions. Inosine and hypoxanthine could not replace inosinic acid, thus demonstrating that the latter was the specific substrate in the enzymatic exchange reaction. 

As mentioned previously, the citrovorum factor had a pronounced effect on the synthesis of inosinic acid de novo from elementary precursors. However, when inosinic acid was included in the reaction medium and bicarbonate omitted (conditions which favored the demonstration of the enzymatic exchange and limited de novo synthesis), it was found that the addition of the citrovorum factor markedly stimulated the incorporation of formate into position 2 of inosinic acid the incorporation of glycine-l-C and of HC OOH into positions 4 and 8, respectively, was only slightly increased. The interpretation of this experiment was that a primary site of action of the citrovorum factor was the incorporation of formate into position 2 of the acid. 

C. Formation of Adenylic and Guanylic Acids from Inosinic Acid. 409... 

The reaction of a one-carbon compound with 5-amino-4-imidazolecar-boxamide ribonucleotide (AICAR) (Fig. 3) completed the purine ring and resulted in the formation of inosinic acid (IMP). AICAR had been postulated earlier as an intermediate in purine syntheris and a stimulatory effect of JV -formyltetrahydrofolic acid (JV -formyl-FH4) on the entry on the one-carbon unit was observed (68). Also, glycine accepted the one-carbon unit from position 2 of IMP, which resulted in the formation of serine and AICAR (143). The reaction was reversible, since AICAR was converted to IMP in the presence of serine (144)- The reaction of AICAR with serine required-TPN and K" ", in addition to a folic acid compound (either Ar5,ATio.anhydroformyl-FH4 or iV -anhydroformyl-FH4) (143). The equation for the reaction is ... 

C. Formation op Adenylic and Guantlic Acids from Inosinic Acid... 

The carbon atoms 2 and 8 in the purine ring of inosinic acid are derived from C1 units. The latter are transferred as activated formate to GAR and AICR as specific formate acceptors. Therefore we have studied the tetra-hydrofolate dependent activation of formate in relation to the netto de novo synthesis of purine nucleotides in cell-free extracts of normal and leukemic leukocytes. In addition, the conversion of exogenous purines to corresponding monophosphoribonucleotides by the specific purine-phosphoribosyItransferases was determined. The aim of these investigations was to study the effect of 6-MP on the formate activating system, which is important for the de novo synthesis of purine nucleotides, on the utilization of preformed purine bases and, in addition, the interaction of allopurinol with 6-MP,... 

In a similar way, enzymes from Azotobacter vinelandii catalyze the transfer of phosphate from adenosine 5-triphosphoric acid and inosine 5-tri-phosphoric acid to the 2-deoxypentonucleoside monophosphates, with the formation of the pyrophosphates and triphosphates. ... 

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