Nucleotide Metabolism: An Introduction

PATERSON

NOMENCLATURE AND ABBREVIATIONS

Enzymes

The 1964 Recommendations of the International Union of Biochemistry are in general followed for the names of enzymes. The Recommended Trivial Names are used in the text. At the ends of most chapters tables give the trivial name, systematic name, and Enzyme Commission number of the principal enzymes discussed. The commonly accepted names of those enzymes not yet listed systematically by the Enzyme Commission have been used.

Purines and Pyrimidines

The structures and numbering systems of the common naturally occurring purine and pyrimidine bases are shown as follows:

Nucleosides and Nucleotides

The terms nucleoside and nucleotide in the strictest sense refer to N-glycosides and phosphorylated N-glycosides, respectively, derived from nucleic acids. The term is now used, however, in several broader ways. Thus, adenosine triphosphate (ATP) is not derived from nucleic acids, but is quite legitimately a nucleotide through its relation to adenosine monophosphate (AMP), which is so derived. Other N-ribosides, such as nicotinamide mononucleotide (NMN), are called nucleotides only by extension and analogy, and nicotinamide-adenine dinucleotide (NAD+), nicotinamide-adenine dinucleotide phosphate (NADP+), etc., are called dinucleotides only by a similar process. Flavin mononucleotide (FMN) is a step still further removed, as it contains ribitol instead of ribose, and flavin-adenine dinucleotide similarly extends the meaning of dinucleotide. N-glycosides such as orotidylate (OMP) and adenylosuccinate are called nucleotides through their close relationship to the true nucleotides.

The terms ribonucleoside, deoxyribonucleoside, etc., will be used in preference to riboside, deoxyriboside, etc.

The basic structures of the ribo- and deoxyribonucleosides and nucleotides are as follows:

Intermediates of Purine Biosynthesis de Novo

The intermediates of the pathway of purine biosynthesis de novo, particularly those which are acyclic, are only called nucleotides by a further extension of the original definition. Although these names are used by tradition, the Enzyme Commission has preferred to regard these compounds as simple phosphoribosyl derivatives. Both old and newer terms are given here; only names based on the Enzyme Commission usage will be used in the text, and they are not abbreviated.

Abbreviations

The IUPAC-IUB Combined Commission on Biochemical Nomenclature has promulgated two sets of abbreviations and symbols for nucleotides and related compounds. While these are suited for description of polynucleotides, the distinction between bases and nucleosides is not always immediately obvious and this distracts from their pedagogical use. The abbreviations used here are, we feel, intuitively obvious and well suited to the portrayal of reaction schemes in which the addition or subtraction of groups occurs.

Ribonucleosides and 2’-Deoxyribonucleosides

Abbreviations for ribonucleosides and 2’-deoxyribonucleosides are derived from those for the bases plus those for the ribosyl or 2’-deoxyribosyl groups. Thus AR stands for adenosine, UR for uridine, and BR for any ribonucleoside; AdR stands for deoxyadenosine, UdR for deoxyuridine, and BdR for any deoxyribonucleoside. When used in abbreviations for nucleotides, N also stands for any ribonucleoside and dN for any deoxyribonucleoside.

Ribonucleoside and 2’-Deoxyribonucleoside Monophosphates

In most cases the traditional abbreviations based on the term nucleoside monophosphate are used. Thus AMP stands for adenosine monophosphates (adenylate), UMP for uridine monophosphate (uridylate), and NMP for any ribonucleoside monophosphate. Similarly, dAMP stands for deoxyadenosine monophosphate (deoxyadenylate), dUMP for deoxyuridine monophosphate (deoxyuridylate), and dNMP for any deoxyribonucleoside monophosphate. To illustrate certain points, the base, sugar, and phosphate group are indicated individually: AMP = ARP; dUMP = UdRP; etc.

Nucleoside Di- and Triphosphates

In most cases the traditional abbreviations based on the terms nucleoside diphosphate (NDP) and nucleoside triphosphate (NTP) are used. Thus ADP stands for adenosine diphosphate and dATP for deoxyadenosine triphosphate. To illustrate certain points, the base, sugar and phosphate groups are indicated individually: ADP = ARPP; dATP = AdRPPP, etc.

Other Terms

PART I

GENERAL ASPECTS OF NUCLEOTIDE METABOLISM

Outline

INTRODUCTION TO GENERAL ASPECTS OF NUCLEOTIDE METABOLISM

Chapter 1: NUCLEOTIDES AS TISSUE CONSTITUENTS

Chapter 2: CONFIGURATION AND CONFORMATION OF NUCLEOSIDES AND NUCLEOTIDES

Chapter 3: FUNCTIONS OF NUCLEOTIDES

Chapter 4: THE THREE LEVELS OF PHOSPHORYLATION

Chapter 5: CARBON AND NITROGEN TRANSFER REACTIONS IN NUCLEOTIDE METABOLISM

Chapter 6: RIBOSE PHOSPHATE SYNTHESIS

INTRODUCTION TO GENERAL ASPECTS OF NUCLEOTIDE METABOLISM

The discovery of a large number of nucleotides as normal tissue constituents, and their separation, isolation, and characterization (Chapter 1), their structures in solution (Chapter 2), and a general discussion of the many and varied functions of nucleotides in cells (Chapter 3), provide a background and general framework for the discussion of the synthesis, interconversion, and catabolism of these natural products which follows in later chapters.

Certain group-transfer reactions are common to many aspects of nucleotide metabolism. These include phosphoryl group transfers which generate the nucleoside polyphosphates found in the cells (Chapter 4), and one-carbon and amino group transfers by which purine and pyrimidine rings are formed and interconverted (Chapter 5). Finally, the formation of the ribose moiety of nucleotides is considered (Chapter 6) to conclude Part I.

CHAPTER 1

NUCLEOTIDES AS TISSUE CONSTITUENTS

Publisher Summary

In 1908, Phoebus Aaron Theodore Levene introduced the term nucleotide to refer to phosphate esters of nucleosides, which were named because they contained a sugar in glycoside linkage with the purine and pyrimidine bases of the nucleic acids. This chapter discusses the historical development of the nucleotide field. The knowledge of the free nucleotides of tissues has developed with the understanding about the chemical nature and metabolism of the nucleic acids. During the 1940s, a new technology was developed for the manipulation and quantitation of microgram quantities of the nucleic acid bases. The advent of this technology, which involved paper chromatography, chromatography on ion-exchange media, and ultraviolet spectro-photometry, brought about a literal explosion in the knowledge of the free nucleotides of cells and tissues. The chapter also discusses nucleoside antibiotics and nucleotide profiles, and the extraction of nucleotides from cells.

I Introduction

II Historical Development

A The Early Period

B The Post-1949 Period

III Free Nucleotides of Cells and Tissues

A The Variety of Free Nucleotides in Nature

B Nucleotide Composition of Cells and Tissues

References

I Introduction

The term nucleotide was introduced in 1908 by Levene to refer to phosphate esters of nucleosides, which were so named because they contained a sugar in glycosidic linkage with the purine and pyrimidine bases of the nucleic acids. The latter term, of course, represents the source from which the bases were first isolated, the nuclei of animal cells.

The purines and pyrimidines are major chemical constituents of cells and occur primarily as components of polymerized nucleotides (nucleic acids) and to a much lesser extent in the form of free (that is, unassociated) nucleotides. Free nucleosides and bases usually represent a very small fraction of the total purine and pyrimidine content of living cells. However, there are exceptions to this generalization, such as the occurrence of substantial amounts of theophylline, theobromine, and caffeine in some plant tissues, and the occurrence of the arabinoside of thymine and uracil in the Caribbean sponge, Cryptotethya crypta. Guanine, as the silvery constituent of fish scales, and the excretion product, uric acid, are free purine products of animal cells, but only occur in substantial amounts extracellularly.

The free nucleotides of cells and tissues are usually isolated from aqueous extracts, the preparation of which involves the precipitation of proteins and nucleic acids with 70% alcohol or with acids (perchloric or trichloroacetic acids are commonly used). The resulting acid-soluble fraction of cells and tissues contains low molecular weight, water-soluble components of the cell, including a complex mixture of free nucleoside phosphates. Modern chromatographic methods with ion-exchange media are able to resolve this mixture, the complexity of which is illustrated in Fig. 1-1; this figure represents the resolution of the mixture of free nucleotides in an acid-soluble extract of mouse liver by chromatography on a column of DEAE-Sephadex. The strong absorption of ultraviolet light in the 250–300 mμ region of the spectrum exhibited by purines, pyrimidines, and their nucleotide derivatives is used in their quantitation (1); this facile and sensitive analytical method has been a critical element in the remarkable expansion of nucleic acid and nucleotide biochemistry in recent times. It is seen in Fig. 1-1 (2) that the mono-, di-, and triphosphates of the ribonucleosides represented in RNA were present in the soluble fraction of liver. The free nucleotide composition of tissues is actually more complex than here indicated because minor nucleotide components, such as the deoxyribonucleoside phosphates, are also present, but remain undetected without the use of special methods. Bases and nucleosides (the two leading peaks) are present, but only in small quantities.

FIG. 1-1 Acid-soluble nucleotides of mouse liver. Rapidly frozen liver tissue was extracted with 0.4 M perchloric acid; the extract was neutralized, chromatographed on a column of DEAE-Sephadex, and the absorbance of eluate fractions at 265 mμ was measured. From (2). Reproduced with permission.

II Historical Development

Our knowledge of the free nucleotides of tissues has developed in step with our understanding of the chemical nature and metabolism of the nucleic acids. A useful perspective may be afforded by the following brief resumé of the historical development of the nucleotide field.

A THE EARLY PERIOD

The nucleic acids were discovered by Miescher in 1868–1869, when he isolated from pus cell nuclei a material which contained phosphorus, was soluble in alkali, but precipitated under acidic conditions. This material was subsequently prepared from other sources and when freed from protein it was called nucleic acid, a term introduced by Altman in 1889. The classical preparations of nucleic acid from yeast yielded a product which we now recognize as ribonucleic acid (RNA). The nucleic acid prepared from thymus glands, thymonucleic acid, was also extensively studied; this material [which, in present terms, was deoxyribonucleic acid (DNA)] was different from yeast nucleic acid. From hydrolysates of these preparations the heterocyclic bases were isolated and characterized. At one time, yeast and thymus nucleic acids were thought to be representative of plant and animal nucleic acids, respectively (3). By 1909, it was apparent that yeast nucleic acid contained adenine, guanine, cytosine, uracil, phosphoric acid, and a sugar which Levene showed at that time to be d-ribose. Thymonucleic acid yielded adenine, guanine, cytosine, thymine, phosphoric acid, and a sugar which was not identified correctly until 1929, when it was characterized as 2-deoxy-d-ribose.

By 1912, ribonucleosides representing the four bases of yeast nucleic acid had been isolated from hydrolysates and their chemical structures determined correctly, except for the improper assignment of the glycosidic linkage to purine N-7. However, chemical hydrolysis did not yield nucleosides from thymonucleic acid and this problem remained unsolved until enzymatic hydrolysis was employed in 1929. Thus, phosphate esters of nucleosides, i.e., nucleotides, were indicated as structural units of nucleic acids. The term nucleotide had been introduced in 1908 by Levene in reference to a hydrolysis product of thymonucleic acid, which he had named thymidylic acid. This compound was recognized as a phosphoric ester of a thymine nucleoside (4). By 1918, the four nucleotide subunits of RNA had been isolated in pure form from alkaline hydrolysates of yeast nucleic acid; however, the position of the phosphate residue was uncertain at that time.

In 1925, the structure of inosinic acid, which was thought to be the 5′-monophosphate of inosine, was confirmed. Inosinic acid, the first free nucleotide to be recognized, has an interesting history (4). In 1847, the barium salt of this substance was isolated from beef extract by Leibig, who derived the name from the Greek words for muscle fiber. The presence of phosphorus in this substance was not recognized until 1895. In 1909, Levene determined the structure of inosine, a hydrolysis product of inosinic acid, to be the riboside of hypoxanthine; with this information, the nucleotide structure of inosinic acid became apparent.

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.

The lability of the sugar component of thymonucleic acid had frustrated attempts to isolate structural subunits by chemical hydrolysis; however, in 1929 a gentle hydrolytic procedure using dog intestinal enzymes yielded the expected four nucleosides, and the sugar was then characterized as 2-deoxy-d-ribose. Thus, it was evident that both types of nucleic acid were polymers of nucleotides. After recognition of DNA in plant tissues and the demonstration of RNA in animal tissues, it was apparent that cells in general contained both types of nucleic acid.

By analogy with muscle adenylate, Levene and Bass (4) anticipated the discovery in tissues of other free nucleotides related to the nucleic acid components. Such nucleotides were indeed found, but their discovery had to await the advent of an adequate technology, which did not become available until after 1949. Prior to the period of recent development in the nucleotide area, several nucleotide coenzymes were discovered:

Nicotinamide adenine dinucleotide phosphate, Warburg et al. (1935)

Nicotinamide adenine dinucleotide, Schlenk and von Euler (1936)

Flavin adenine dinucleotide, Warburg and Christian (1938)

Coenzyme A, Lipmann (1946)

Prior to 1936, the purine nucleotides were formulated as having the pentose at N-7 because of an arbitrary assignment to this position by Fischer. In 1936, Gulland was able to assign the glycosidic bond to N-9 on the basis of comparisons of absorption spectra of synthetic N-7 and N-9 substituted purines. During the 1940’s, the chemistry of the nucleosides was developed extensively and the furanose nature of the sugar was established, as was the β-configuration of the glycosyl linkages (5).

B THE POST-1949 PERIOD

During the 1940’s, a new technology was developed for the manipulation and quantitation of microgram quantities of the nucleic acid bases. The advent of this technology, which involved paper chromatography, chromatography on ion-exchange media, and ultraviolet spectrophotometry, brought about a literal explosion in our knowledge of the free nucleotides of cells and tissues.

In 1949, Cohn applied ion-exchange chromatography to the separation of nucleic acid derivatives and demonstrated elegant separations of nucleotides on anion-exchange resins such as Dowex-1 (which has quaternary ammonium charged groups on a polystyrene matrix). It was shown at that time that both RNA and DNA could be hydrolyzed by phosphodiesterases to yield nucleoside 5′-phosphates and it was therefore evident that polynucleotides could be regarded as assemblies of nucleoside 5′-phosphates. At this point, the adenosine phosphates were the only free nucleotides known to occur in tissues, apart from the coenzymes. Because the free adenosine phosphates were 5′-esters, a precursor relationship between these compounds and the polynucleotides seemed likely.

A major step was taken by V. R. Potter and his associates in experiments which, for the first time, applied anion-exchange chromatography to the isolation of nucleotides from the acid-soluble fractions of liver and other tissues. A complex mixture of free nucleotides was at once shown to be present in tissues (see Fig. 1-2). The 5′-mono-, di-, and triphosphates of the four ribonucleosides present in RNA were identified, as were several nucleotide conjugates (6–8). Deoxyribonucleoside phosphates were not evident in these studies, but were found subsequently by others.

FIG. 1-2 Acid-soluble nucleotides of the Flexner-Jobling rat carcinoma. Pooled tumor tissue was extracted with 0.4 M perchloric acid; the neutralized extract was chromatographed on a column of Dowex-1 resin (formate form) using gradient elution with formic acid-ammonium formate solutions. From (8). Reproduced with permission.

Free deoxyribonucleotides are evidently formed in cells at the time of DNA synthesis (9) and are not present in appreciable quantities in nonproliferating cells. By rechromatographing nucleotide-containing fractions isolated from calf thymus extracts by anion-exchange chromatography, R. L. Potter and co-workers (10) were able to isolate the mono-, di-, and triphosphates of deoxycytidine and thymidine. dATP was isolated from a transplantable rat tumor by LePage (11). It became evident that only very small quantities of free deoxyribonucleosidic materials were present in animal tissues; several indirect methods of high sensitivity have been developed to meet this analytical problem. Deoxyribonucleosidic substances in tissue extracts have been measured by highly sensitive microbial assays using lactobacilli with growth requirements for deoxyribonucleosides (12, 13). Pool sizes of the deoxyribonucleotides have also been measured by an indirect method in which cells were cultured in media containing ³²P-phosphate of known specific activity; the acid-soluble nucleotides extracted from such cells were determined by means of the radioactivity which co-chromatographed with samples of authentic deoxyribonucleotides (14). By another indirect method employing DNA polymerase, pool sizes of dATP, dGTP, dCTP, and dTTP have been measured in cultures of mouse embryo cells (15, 16).

With the advent of an adequate nucleotide technology and the development of widespread interest in nucleotide metabolism, many free nucleotides have been found in cells; however, no comprehensive survey has appeared since the 1958 report of Henderson and LePage (17), which lists over 100 nucleotides, and the list has probably more than doubled since. A large number of free nucleoside diphosphate conjugates have been isolated from cells and characterized; many such compounds serve as coenzymes in group-transfer reactions, for example, CDP-X and UDP-X derivatives participate, respectively, in phospholipid and polysaccharide synthesis (see chapter 3). As well, many new nucleotides have been recognized as metabolites of nucleoside antibiotics and of synthetic purine and pyrimidine