Adenylic acid, deamination

The bearing which these discoveries have had on the elucidation of the structure of ribopolynucleotides will be discussed later. It is important to stress here, however, that, for most purposes, the older methods of preparing nucleotides have been superseded by procedures which yield separate isomers of each. Of the techniques mentioned above, paper chromatography iB mainly of analytical value, and is the most convenient method for the qualitative detection of isomeric adenylic acids. The only disadvantage of this method is that the isomers are not completely separable from muscle adenylic acid. The presence of the latter, however, can be readily detected by hydrolyzing it to adenosine by means of the specific 5-nucleotidase present in snake venoms,66 or by deamination by a specific enzyme... 

The isomerism existing between the pairs of nucleotides was attributed to the different locations of the phosphoryl residues in the carbohydrate part of the parent nucleoside,49 63 since, for instance, the isomeric adenylic acids are both hydrolyzed by acids to adenine, and by alkalis or kidney phosphatase to adenosine. Neither is identical with adenosine 5-phosphate since they are not deaminated by adenylic-acid deaminase,68 60 and are both more labile to acids than is muscle adenylic acid. An alternative explanation of the isomerism was put forward by Doherty.61 He was able, by a process of transglycosidation, to convert adenylic acids a" and 6 to benzyl D-riboside phosphates which were then hydrogenated to optically inactive ribitol phosphates. He concluded from this that both isomers are 3-phosphates and that the isomerism is due to different configurations at the anomeric position. This evidence is, however, open to the same criticism detailed above in connection with the work of Levene and coworkers. Further work has amply justified the original conclusion regarding the nature of the isomerism, since it has been found that, in all four cases, a and 6 isomers give rise to the same nucleoside on enzymic hydrolysis.62 62 63 It was therefore evident that the isomeric nucleotides are 2- and 3-phosphates, since they are demonstrably different from the known 5-phosphates. The decision as to which of the pair is the 2- and which the 3-phosphate proved to be a difficult one. The problem is complicated by the fact that the a and b" nucleotides are readily interconvertible.64,64... 

Although adenylic acid deaminase is a well-known enzyme that has been isolated in crystalline form, little work has been reported on its substrate specificity evidence for the deamination of 4-aminopyrazolo[3,4-d]pyrimidine ribonucleotide has been presented [118], but it is not known if it catabolizes any of the other intracellularly formed adenylic acid analogues. 

The existence of two separate enzymes in animal tissues responsible for the liberation of ammonia from each of the two aminopurines, adenine and guanine, the latter specific for the free purine and the former for the nucleosides, was initially presented by Jones and his colleagues 11, 12). In 1928, Schmidt 13-15) demonstrated that AMP aminohy-drolase was responsible for the appearance of inosinic acid in muscle and for at least a portion of ammonia liberated during contraction. He showed not only a marked specificity for deamination of 5 -AMP but also provided the first clue that muscle adenylic acid (5 -AMP) and yeast adenylic acid (3 -AMP) were different compounds. Initial evidence for guanine and adenosine aminohydrolase including aspects of the specificity were also described by Schmidt 16). Additional details regarding development of interest in purine aminohydrolases are available in several excellent reviews 17-20). 

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). 

Adenylic deaminase. The deamination of 5 -adenylic acid by 5 -adenylic deaminase results in the formation of 5 -inosinic acid. This process, applied to mushrooms, intensifies the natural flavor (53). 

The enzyme adenylic acid deaminase catalyzes the deamination of AMP to IMP and ammonia. For the HPLC method, the assay involves the separation of the substrate, AMP, from the reaction product IMP. The enzyme is found in muscle. 

Scheme 10.15 Hydrolytic deamination of ADA and AMPDA of adenosine and adenylic acid.

Inosinic and Adenylic Adds, Similarly, Levene and Harris found that the adenylic acid from ribosenucleic acid can be deaminated to an inosinic acid which undergoes hydrolysis at its own pH to give hypo-xanthine and 3-phosphoribose. On the other hand, Thannhauser had shown that ammoniacal hydrolysis of the adenylic acid gives adenosine and phosphoric acid. 

Derivation From meat extract or by enzymatic deamination of muscle adenylic acid. 

Oxidation of (8) yielded a ribonic acid phosphate (9), whose properties differed from those of the ribonic acid 5-phosphate obtained by similar hydrolysis and oxidation of inosine 5 -phosphate. Moreover, reduction of (8) allegedly gave a ribitol phosphate (10) which was optically inactive. From these studies, it followed that the phosphoric moiety in (10) is esterified by the 3-hydroxyl group of the ribitol moiety. Similarly, a yeast adenylic acid, obtained from an alkaline hydrolysate of yeast ribonucleic acid, was deaminated to an inosinic acid that was different... 

Crystals from water + acetone, mp 196-200". [alff -47,5 (c = 2, 2% NaOH) -26.0 (c = 2, 10% HCI). pK, -- 3.8 pKj — 6.2. aM (molar absorbancy) 15.4 X I03 at 259 nm (pH 7.0). Readily sol in boiling water. The compound is readily deaminated by nitrous acid to form inosinic acid less rapidly hydrolyzed than 3 -adenylic acid by sulfuric acid. Furfural is formed only in traces On distillation with 20% HCI, cf. Levene, Bass, Nucleic Acids (New York, 1931) pp 230-232. Absorption spectrum Kalckar. foe. cir. thERap cat-. Nutrient. 

Japan, pat. 732C86) (to Ajinomoto), C.A. 51, 3870b (1957). Structure Levene, Bess, op. ctt.. pp 187-192 Bredereck, Ber. 66, 198 (1933) Levene, Tipson, J. Biol. Chem. Ill, 3t3 (1935). Also prepd from muscle by enzymatic deamination of muscle adenylic acid Ostem, Biochem. Z. 254, 63 (1932) by hydrolysis of inosine triphosphate Kleinzeller, Biochem. J. 36, 729 (1942). Studies on the enzymatic synthesis Greenberg, J. Biol. Chem. 198, 611 (1951) Korn et al, ibid 217, 875 (1955). Microbial fermentation method using mutant strains of Micrococcus glutamicus Kinoshita el al. U.S. pat. 3,232,844 (1966 to Kyowa). 

An important discovery, that of free adenylic acid in muscle, was made by Embden in 1927. Muscle adenylate was recognized as the 5 -mono-phosphoric ester of adenosine because enzymatic deamination yielded the known inosinic acid. It was shown at that time that the deaminase preparations from muscle did not deaminate the adenylic acid isolated from alkaline hydrolysates of yeast nucleic acid as well, differences were apparent in the chemical properties of the adenylic acids from these two sources. Yeast adenylic acid and the other nucleotides from alkaline hydrolysates of RNA were ultimately shown to be mixtures of the 2 - and 3 -phospho esters. In 1929 the isolation of adenosine triphosphate from muscle was reported by Lohmann and independently by Fiske and Subbarow. The discovery of adenosine diphosphate followed in 1935. 

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. 

Mammalian tissues do not contain adenine deaminase, but specific enzymes able to deaminate adenosine and 5 -adenylic acid have been found in a variety of mammals. Adenosine deaminase activity has been detected in intestine, liver, spleen, brain, kidney, heart, and skeletal muscle. Adenosine deaminase has been partially purified from the intestinal mucosa. The enzyme has a great affinity for adenosine and deoxyadenosine and only low affinity for 2 -AMP and 3, 3 -cyclic AMP. Its activity is lost on dialysis. The enzyme acts optimally at pH 7. 

This finding by Kalckar and Rittenberg of a rapid turnover of the amino group of ATP suggests that resting muscle is subject in vivo to deamination-reamination processes. This is corroborated by earlier evidence obtained during severe muscular work, in which adenylic acid... 

With the activities of different enzymes in mind, a rapid enzymatic deamination of adenosine and adenylic acid before the compounds per se could be utilized for nucleic acid synthesis could explain the less efficient incorporation of these substances " (in contrast to adenine), since adenosine deaminase and adenylic acid deaminase have been repeatedly demonstrated in tissues. " "... 

The possibility existed that adenylic acid might have been the first purine compound synthesized by the homogenate, and that the isolated inosinic acid was merely a deamination product of adenylic acid. However, labeled formate was not incorporated into the adenine nucleotides of the homogenate or into added adenylic acid. 

Pentose nucleic acids are degraded by various enzyme systems to yield mononucleotides, and the resulting purine compounds are converted stepwise to uric acid. For example, the purine mononucleotide, adenyhc acid (adenosine-5 -phosphate), can be hydrolytically deaminated by 5-adenylic acid deaminase to yield inosinic acid, or it can be directly... 

Some common examples of enzymes inhibited by phosphate ions include carboxypeptidase, fumarase, urease, phos-phoglucomutase, carboxylase, arylsulphatase and muscle deaminase (for the deamination of adenylic acid). Frequently this inhibition is due to competition of the phosphate with substrates containing phosphate groups or to complex formation with a metal ion essential for the enzyme activity. 

Evidence exists for specific enzymes which will deaminate 3-adenylic acid, 5-adenylic acid, guanylic acid, adenosine, guanosine, and cytidine. Little is known concerning their distribution. 

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. 

In the second experiment, the rapid degradation of AMP by pigeon liver was mediated almost entirely by phosphatase action rather than by deamination (67). Since the specific activity of IMP formed from small precursors in incubated homogenates was unaffected by the simultaneous breakdown of adenylic acid, it was concluded that adenosine, rather than IMP, was the first product of AMP catabolism. If IMP were formed from AMP the specific activity of IMP would have been lowered. 

The 5 -adenyhc acid deaminase (22) found in rabbit muscle has been crystallized (23). It converts adenylic acid to inosinic acid and ammonia [Eq. (7)]. The enzyme does not deaminate adenine, adenosine, adenosine diphosphate, adenosine triphosphate, adenosine 2 -phosphate, adenosine S -phosphate, guanosine, or cytosine but does act upon deoxyadenylic acid (24)-... 

Ammonia Carriers.— Although ammonia appears as an end-product of general deamination, it is too toxic and reactive a metabolite to remain in the tissues in a free form, and is taken up by appropriate carriers, such as glutamic acid, aspartic acid, adenylic acid and adenosine, or may be removed by an amino-pherase system. These, or other carriers, transport ammonia to the liver for detoxication by conversion to urea. 

Adenylic acid, now called muscle adenylic acid to distinguish it from adenylic acid obtained from yeast, is widely distributed in animal tissue, and ranks along with histamine and acetyl choline as a powerful vaso-dilator. On deamination it is converted to the much less active inosinic acid. Adenylic diphosphate (or pyrophosphate) is found in muscle where it acts as a donator of phosphoric acid in the contraction process (p. 291), becoming degraded to adenyl phosphate durii the change. 

According to Conway (1938), adenosine and adenylic acid are the chief sources of the ammonia latent in blood, and are deaminated by plasma and tissue deaminases, with less of the free amino group at position (6). 

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