Pyrimidine nucleotide hydrolysis

In view of the difficulty of hydrolyzing the pyrimidine nucleosidic linkages, ribonucleic acids have been hydrolyzed to a mixture of purine bases and pyrimidine nucleotides which is then separated by paper chromatography.132, 163 164 This method has been employed extensively for the analysis of ribonucleic acids, and gives reproducible results,166 but it has not been used to any great extent for deoxyribonucleic acids, probably because, under these conditions of hydrolysis, they yield some pyrimidine deoxy-ribonucleoside diphosphates.166... 

Enzymes which catalyze the hydrolysis of the unit linkage of sequential residues of oligomers or polymers determine their substrate specificity by recognizing the particular unit residue in the sequential chain as well as the direction of the chain. For example, ribonuclease cleaves the 3 -phosphate of a pyrimidine nucleotide residue but not the 5 -phosphate, and trypsin hydrolyzes peptide bonds which involve the arginine or lysine residue at the carbonyl end but not at the amino end. This is also the case for the hydrolysis of a variety of synthetic substrates and quasi-substrates (Sect. 4.1). Synthetic trypsin substrates are ester or amide derivatives in which the site-specific group (positive charge) is contained in their carbonyl portion. 

In de novo synthesis of pyrimidines, the ring is synthesized first and then it is attached to ribose to form a pyrimidine nucleotide (Figure 25.2). Pyrimidine rings are assembled from bicarbonate, aspartic acid, and ammonia. Although ammonia can be used directly, it is usually produced from the hydrolysis of the side chain of glutamine. 

At this stage, orotate couples to ribose, in the form of 5-phosphoribosyl-l-pyrophosphate (PRPP), a form of ribose activated to accept nucleotide bases. PRPP is synthesized from ribose-5-phosphate, formed by the pentose phosphate pathway, by the addition of pyrophosphate from ATP. Orotate reacts with PRPP to form orotidylate, a pyrimidine nucleotide. This reaction is driven by the hydrolysis of pyrophosphate. The enzyme that catalyzes this addition, pyrimidine phosphoribosyltransferase, is homologous to a number of other phosphoribosyltransferases that add different groups to PRPP to form the other nucleotides. Orotidylate is then decarboxylated to form uridylate (IMP), a major pyrimidine nucleotide that is a precursor to RNA. This reaction is catalyzed by orotidylate decarboxylase. 

Levene discovered that, on hydrolyzing ribosenucleic acid with boiling 2% sulfuric acid under reflux during two hours, the purine nucleotides are decomposed but the two pyrimidine nucleotides, namely uridylic and cytidylic acids, are resistant to this treatment. He succeeded in separating the mixture of pyrimidine nucleotides, through the barium salts, into crystalline barium uridylate and amorphous barium cytidylate, in the same year that Thannhauser crystallized brucine uridylate. Finally, in 1920, Levene reported the crystallization of the free pyrimidine nucleotides. On neutral hydrolysis they yield uridine and cytidine, respectively. 

Bolomey and Allen found that a non-specific phosphatase preparation (Bredereck ) containing a small amount of ribonuclease hydrolyzes ribosenucleic acid in such a manner that guanosine is liberated faster than adenosine, in the early stages of the hydrolysis the equivalent amount of free phosphoric acid is simultaneously formed. After hydrolysis of the purine nucleotide constituents has reached a maximum, hydrolysis of the pyrimidine nucleotides becomes appreciable. (If the ribosenucleic acid is subjected to the action of ribonuclease before treatment with the phosphatase, the reaction is much more rapid.) They therefore tentatively suggested that guanylic acid is the first mononucleotide liberated and adenylic acid the second. Hence, provided that... 

It would be of interest to halt the hydrolysis when liberation of guanosine and adenosine approaches a maximum, and determine whether the pyrimidine nucleotides are present as a dinucleotide or as the two mononucleotides. It is not clear whether the action of the non-specific phosphatase on an artificially-prepared, equimolecular mixture of the four mononucleotides has been studied (although the individual mononucleotides have been so examined by Bredereck, Beuchelt and Richter ), but Kobayashi has found that guanylic acid is hydrolyzed more readily than adenylic acid which, in turn, is hydrolyzed more readily than the pyrimidine nucleotides. Furthermore, Bredereck, et oZ. have shown that mild chemical hydrolysis of ribosenucleic acid with aqueous pyridine at 100° gives guanylic acid (G) plus a trinucleotide composed of adenylic (A), cytidylic (C), and uridylic (U) acids. On further hydrolysis in aqueous pyridine, adenylic acid is split off. Hence, in ribosenucleic acid, (G) is at one end of the molecule and, in the trinucleotide, (A) is at one end of the molecule. Possible formulas for the tetranucleotide are therefore... 

Desoxyribosenucleic acid is readily hydrolyzed by mineral acids but is more resistant to alkaline fission than is ribosenucleic acid. Owing to the nature of the constituent sugar, the purine nucleotides are even more unstable than those of ribosenucleic acid. Hence, by acid hydrolysis of thymus nucleic acid, only the two pyrimidine nucleotides" " can be isolated. 

As previously mentioned, Levene was able to isolate the pyrimidine nucleotides of desoxyribosenucleic acid by chemical hydrolysis but, owing to their instability, the purine nucleotides were destroyed. 

The natural function of the carboxymethylhydantoinase (E. C. 3.5.2.2) is postulated to be the hydrolysis of 5-carboxymethylhydantoin, which is described to be the product of a non-enzymatic cyclization of N-carbamoyl-i-aspartic acid123, 241 and to occur as a side-product in the metabolism of the pyrimidine nucleotide dihydroorotic acid1251. This enzyme often occurs in combination with a ureidosuccinase (E.C. 3.5.1.7)[2S1, which catalyzes the cleavage of the resulting N-carbamoyl aspartic acid to L-aspartic acid (see Fig. 12.4-5). L-5-Carboxymethylhydantoin was first isolated after incubating orotic acid, a six-membered cyclic amide, with crude cell extracts of the anaerobic bacterium Clostridium oroticum125, 261. 

The first step in de novo pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from bicarbonate and ammonia in a multistep process, requiring the cleavage of two molecules of ATP. This reaction is catalyzed by carbamoyl phosphate synthetase (CPS), and the bicarbonate is phosphorylated by ATP to form carboxyphosphate and ADP (adenine dinucleotide phosphate). Ammonia then reacts with carboxyphosphate to form carbamic acid. The latter is phosphorylated by another molecule of ATP with the mediation of CPS to form carbamoyl phosphate, which reacts with aspartate by aspartate transcarbamoy-lase to form A-carbamoylaspartate. The latter cyclizes to form dihydroorotate, which is then oxidized by NAD-1- to generate orotate. Reaction of orotate with 5-phosphoribosyl-l-pyrophosphate (PRPP), catalyzed by pyrimidine PT, forms the pyrimidine nucleotide orotidylate. This reaction is driven by the hydrolysis of pyrophosphate. Decarboxylatin of orotidylate, catalyzed by orotidylate decarboxylase, forms uridylate (uridine-5 -monophosphate, UMP), a major pyrimidine nucleotide that is a precursor of RNA (Figure 6.53). 

During digestive processes, nucleoprotein is split into nucleic acids and protein, the latter then being broken down into amino acids. The nucleic acids are attacked by ribonuclease and deoxyribonuclease enzymes to form nucleotides, which are further hydrolysed by nucleotidases to form nucleosides and phosphates. In the intestines these nucleosides are split by nucleosidases into ribose, deoxy-ribose, purine and pyrimidine bases, which later undergo oxidation and decomposition to ammonia, carbon dioxide and water, to be finally expelled as urea. Nucleotide hydrolysis products are conveniently identified and isolated by chromatographic methods (Chapter 14.2). 

The separation of nucleotides and deoxynucleotides, previously a formidable task involving the fractional crystallization of heavy metal and alkaloid salts 102) has been made much easier by developments in analytical techniques. Ion-exchange methods may be used for the purification, isolation, and identification of both classes of nucleotides from hydrolysis mixtures 103), Countercurrent distribution 104) and starch 106) and cellulose-column 106) as well as paper-strip chromatography 107) have also proved to be useful in separating nucleotides from natural sources. Spectro-photometric procedures based on the characteristic ultraviolet absorption spectra of the purines and pyrimidines have been the most convenient method to locate, estimate, and identify the fractions obtained in the previous separations. Since the nucleotides are acid in nature, they are often named as acids, e.g., adenylic acid, cytidylic acid. The general constitution of the purine nucleotides (and by analogy the pyrimidine nucleotides) is demonstrated by their hydrolysis by acids to a purine and ribose (or 2-deoxyribose) monophosphate and by alkalies to the nucleosides and phosphoric acid. The order of the constituents in a purine nucleotide must, therefore, be ... 

Hydrolysis of RNA by crystalline pancreatic ribonuclease likewise proceeds through intermediate 2, 3 -cyclization 164)y but in this case the action is specifically limited to phosphoryl linkages associated with the pyrimidine nucleotides the cyclic intermediates subsequently are degraded by the enzyme only to the 3 -nucleotide type. Thus, the end-products are polynucleotides which terminate in 3 -pyrimidine nucleotide groups, and pyrimidine mononucleotides of the 3 -variety 165). The structural identification of many of the polynucleotides demonstrated that no simple alternating sequence of purines and pyrimidines exist in the intact RNA. 

Pancreatic ribonuclease. Ribonuclease (MW 13,600), with known amino-acid sequence and tertiary structure, is quite heat stable and frequently a great nuisance, because of its ubiquitous presence and Its stability. It attacks only single-stranded RNA and cleaves at the 5 -side of pyrimidine nucleotides (see Fig. 3.8). The reaction, therefore, yields oligonucleotides with -Yp, I.e., with the 3 -phosphate group on the terminal pyrimidine nucleoside. Its reaction mechanism is well known and closely follows that of alkaline hydrolysis, i.e., the intermediate is a pyrimidine nucleoside-2, 3 -cycllc phosphate. 

The nucleic acids DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are biological polymers that act as chemical carriers of an organism s genetic information. Enzyme-catalyzed hydrolysis of nucleic acids yields nucleotides, the monomer units from which RNA and DNA are constructed. Further enzyme-catalyzed hydrolysis of the nucleotides yields nucleosides plus phosphate. Nucleosides, in turn, consist of a purine or pyrimidine base linked to Cl of an aldopentose sugar—ribose in RNA and 2-deoxyribose in DNA. The nucleotides are joined by phosphate links between the 5 phosphate of one nucleotide and the 3 hydroxyl on the sugar of another nucleotide. 

Alkaline hydrolysis splits the nucleotide into its phosphate and sugar-base residues. The sugar-base is known as a nucleoside. The nucleosides are named according to the type of base present. If a purine base is present it will end -osine, e.g. adenosine, while if a pyrimidine is present the name will end -idine, e.g. uridine. 

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