Purine bases inosinic acid

Robert Holley first determined the base sequence of a tRNA molecule in 1965, as the culmination ul 7 years of effort, Indeed, his study of yeast alanyl-tRNA provided the first complete sequence of any nucleic acid. This adapter molecule is a single chain of 76 ribonucleotides (Figure 30.2). The 5 terminus is phosphorylated (pCi), whereas the 3 terminus has a free hydroxyl group. T he amino acid-attachment site is the 3 -hydroxyl group of the adenosine residue at the 3 terminus of the molecule. The sequence 5 - IGC-3 in the middle of the molecule is the anticodon, where I is the purine base inosine. It is complementary to 5 -GCC-3, one of the codons for alanine. 

In the most important degradative pathway for adenosine monophosphate (AMP), it is the nucleotide that deaminated, and inosine monophosphate (IMP) arises. In the same way as in GMP, the purine base hypoxanthine is released from IMP. A single enzyme, xanthine oxidase [3], then both converts hypoxanthine into xanthine and xanthine into uric acid. An 0X0 group is introduced into the substrate in each of these reaction steps. The oxo group is derived from molecular oxygen another reaction product is hydrogen peroxide (H2O2), which is toxic and has to be removed by peroxidases. 

Adenosine deaminase (ADA) is a ubiquitous enzyme that is essential for the breakdown of the purine base adenosine, from both food intake and the turnover of nucleic acids. ADA hydrolyzes adenosine and deoxyadenosine into inosine and deoxyinosine, respectively, via the removal of an amino group. Deficiency of the ADA enzyme results in the build-up of deoxyadenosine and deoxyATP (adenosine triphosphate), both of which inhibit the normal maturation and survival of lymphocytes. Most importantly, these metabolites affect the ability of T-cells to differentiate into mature T-cells [656430], [666686]. ADA deficiency results in a form of severe combined immunodeficiency (SCID), known as ADA-SCID [467343]. 

Purine nucleotides are degraded by a pathway in which they lose their phosphate through the action of 5 -nucleotidase (Fig. 22-45). Adenylate yields adenosine, which is deaminated to inosine by adenosine deaminase, and inosine is hydrolyzed to hypoxanthine (its purine base) and D-ribose. Hypoxanthine is oxidized successively to xanthine and then uric acid by xanthine oxidase, a flavoenzyme with an atom of molybdenum and four iron-sulfur centers in its prosthetic group. Molecular oxygen is the electron acceptor in this complex reaction. 

The purine bases, adenine and guanine, participate in nature by providing structural elements of nucleic acids and numerous cofactors. The deaminated products of adenine and guanine do not participate in these coenzymic functions except in a relatively few instances. For example, the relative ineffectiveness of inosine monophosphate (IMP) vs. 

The phosphodiester bonds of xanthylic acid in deaminated RNA were scarcely split by RNase U2 (30). The susceptibility of purine nucleotide residues to RNase U2 decreases in the order of A>G>I X, indicating that the phosphodiester bonds of adenylic acid and inosinic acid without a keto group at the position of purine base are more sensitive to RNase U2 than those of guanylic acid and xanthylic acid. The resistance of TNP-RNA to RNase U2 may be also attributed to the steric hindrance by a larger substituent at 2-amino groups of guanylyl residues, as with RNase T, (SO). 

Purine nucleotides can be produced by two different pathways. The salvage pathway utilizes free purine bases and converts them to their respective ribonucleotides by appropriate phosphoribosyltransferases. The de novo pathway utilizes glutamine, glycine, aspartate, N -formyl FH4, bicarbonate, and PRPP in the synthesis of inosinic acid (IMP), which is then converted to AMP and GMP. 

Two of the more important purine analogs in use clinically are 6-mercaptopurine and 6-thioguanine. These purine antagonists and glutamine antagonists such as azaserine (Table 4-6) are major antagonists in the biosynthesis of purine bases. Before understanding the mechanism of their action, it is necessary to look at the biosynthesis of inosinic acid, the purine ribonucleotide that is the precursor to both purine bases found in DNA and RNA... 

Hypoxanthine is a base found in an intermediate of purine nucleotide biosynthesis. Figure 22.4 summarizes the pathway leading from phosphoribosyl-1-pyrophosphate (PRPP) to the first fully formed purine nucleotide, inosine 5 -monophosphate (IMP), also called inosinic acid. IMP contains as its base, hypoxanthine. 

Mild acid hydrolysis of the deaminated purine nucleotides, xanthylic acid and inosinic acid, gives the purine bases (xanthine and hypo-xanthine, respectively) and a reducing sugar phosphate. The same hydrolytic products are obtained by use of a specific pancreatic enzyme. On the other hand, mild alkaline hydrolysis of a nucleotide, or treatment with the appropriate enzyme, liberates free phosphoric acid and a non-reducing compound of base and sugar, known as a nucleoside. (Hydrolysis of ribosenucleic acid with fairly dilute ammonia under pressure, during 3.5 hours at a bath temperature of 175 to 180 , gives an equimolecular mixture of four nucleosides). 

In the case of DNA, a D-2-deoxyribose molecule is combined to each of the bases to form a nucleoside, and the nucleosides are then combined with each other with a phosphoric acid to form a polymer (DNA). On the other hand, in the case of RNA, D-ribose, instead of D-2-deoxyribose, is combined to each of the bases to form a nucleoside, and as in the case of DNA, these nucleosides are combined with each other to form a polymer (RNA). Among the bases within DNA and RNA, adenine and guanine have been described in the preceding section. In this section, cytosine, thymine, and uracil, which are pyrimidine bases, will be described. Purine derivatives exist as a constituent unit of nucleic acids and as many kinds of monomers, and these are also present in natural products, such as caffeine, inosinic acid, and cytokinin. On the other hand, as natural products, pyrimidine derivatives are rather rare. Nucleosides composed of pyrimidine bases cytosine, thymine, and uracil coupled with D-ribose are known as cytidine, thymidine, and uridine, respectively. Among these alkaloids, cytidine was first isolated from the nucleic acid of yeast [1,2], and thymidine was isolated from thymonucleic acid [3,4]. In the meantime, uridine was obtained by the weak alkali hydrolysis [5] of the nucleic acids originating from yeast. 

As described in the previous section, in the biosynthesis of inosinic acid, ribose combines with the purine base in the initial stages of purine skeleton synthesis. On the other hand, in the biosynthesis of the pyrimidine nucleosides, ribose is introduced after the completion of the synthesis of the... 

The base composition of tRNA is different from that of ribosomal and nuclear RNA. One of the most striking differences is the presence of rare bases, such as pseudouridine, thiouridine, dihydrouridine, methyl purines, and inosine. The significance of this is not clear. These bases are assumed to play a determinant role in amino acid selection. Yet Littauer and his associates [151] found no differences between the abilities of methylated and unmethylated transfer RNA to accept leucine, methionine, and isoleucine. Unmethylated RNA, however, is more sensitive to RNase than methylated RNA. Each tRNA almost certainly does not contain all the odd bases. Pseudouridine is the only one that has been found consistently in all the RNA s studied. 

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. 

The fate of other purine-ribose compounds was studied in the rat and it was found that C Mabeled adenosine (211) and adenylic acid (212) were utilized for the s3Tithesis of RNA adenine and guanine, but to a much smaller extent than adenine (191). Similarly, growing yeast utilized the purine base, adenine, far more readily than the corresponding nucleoside or nucleotide (195). It was believed that the ribose derivatives were poorly utilized because they were first cleaved to free adenine, which was incorporated subsequently into polynucleotides. It is curious that the attachment of ribose or a ribose pho hate moiety to adenine or guanine did not facilitate their incorporation into nucleic acids. In contrast, inosine, the ribonucleoside of hypoxantbine, was utilized considerably by the rat as a nucleic acid precursor (211) the corresponding deoxyriboside, deoxyinosine, was not (213). 

There are several pathways available for the degradation of the mononucleotides. For example, adenosine 5 -phosphatc (AMP) is either deaminated hydrolytically to inosinic acid (IMP) by 6 -adenylic acid deaminase (217, iS7) or split directly to the corresponding nucleoside, adenosine, by 5 -nucleotidase 238). The nucleoside inosine resulted from either the hydrolysis of inosinic acid by 5 -nucleotidase or by the action of adenosine deaminase on adenosine 238, 239). The above pathways, as well as other likely conversions of purine compounds to hypoxanthine and xanthine 2JiO) are shown in Fig. 13. Finally, the enzyme xanthine oxidase acted on both the free bases, hypoxanthine and xanthine, to produce uric acid which was the final product of purine metabolism in some animals. 

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,... 

The Lesch-Nyhan syndrome is a rare, X-linked genetic disease due to a functional absence of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (Seegmiller, Rosenbloom and Kelley, 1967). This enzyme catalyzes the transfer of the 5-phos-phoribosyl moiety of 5-phosphoribosyl-l-pyrophosphate (PP-ribose-P) to the purine bases guanine and hypoxanthine to form the nucleotides inosinic acid and guanylic acid. 

Autoradiography with labeled substrates on cultured fibroblasts has been used to visualize HG-PRT deficiency cells of deficient individuals lack the ability to incorporate hypoxanthine or guanine into nucleic acids, because these purine bases can not be converted to their corresponding mononucleotides (1,2). The alternative pathway to form IMP or GMP via inosine or guanosine is not likely, for, although nucleoside phosphorylase is present, there is no definite evidence for the existence of inosine- or guanosine kinase in human cells (3,4). 

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