BIOCHEMISTRY AND MOLECULAR BIOLOGY - W. ELLIOTT - 2002

CHAPTER 18. NUCLEOTIDE METABOLISM

In several preceding chapters, the primary focus was on energy generation from nutrients. ATP plays a central role in this process, although GTP, CTP, and UTP are also involved in various metabolic reactions. However, until now we have mainly considered the phosphate groups of these molecules. The Nature of the bases—whether A, G, C, or U—was important only for their recognition by the corresponding Enzymes and had no direct bearing on metabolic processes. For this reason, we did not previously discuss The Structure of the bases themselves.

In this chapter, we move on to a major new area of biochemistry: The Study of information transfer mechanisms. We will examine Nucleic Acids and The process of Protein Synthesis, which requires a knowledge of the STRUCTURE OF THE basic NUCLEOTIDES. This chapter is dedicated to the structure, synthesis, and METABOLISM of nucleotides and serves as essential Background for understanding the subsequent chapters.

STRUCTURE AND NOMENCLATURE of Nucleotides

The term nucleotide originates from nucleic acids, which were first discovered in the Cell Nucleus (Lat. nucleus) and consist of polymers of nucleotides. Nucleotides share a common structure: phosphate-sugar-base.

The structure of nucleosides can be written as sugar-base.

Thus, AMP and the corresponding nucleoside, adenosine, have the following structure:

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Strictly speaking, AMP is written here as 5'-AMP. The prime (') indicates that the numbering refers to the atoms in the ribose sugar ring rather than the adenine ring. However, the 5'-position of the phosphate, being the most common, is usually omitted. Therefore, 5'-AMP is often simply called AMP, whereas the phosphate position at the third carbon atom of ribose is explicitly designated as 3'-AMP.

Carbohydrate Component of Nucleotides

The carbohydrate component of nucleotides is always a pentose—most commonly ribose or 2'-deoxyribose—which invariably has the D-configuration and is never found in the L-form.

In RNA, the carbohydrate is always ribose (hence the name ribonucleic acid), whereas in DNA it is deoxyribose (hence deoxyribonucleic acid). A nucleotide containing ribose is a ribonucleotide, but this is usually not specified in writing; unless otherwise indicated, a nucleotide denoted as AMP refers to ribonucleotides. When designating deoxyribonucleotides, either the prefix deoxy- is used (e.g., deoxyadenosine monophosphate) or a "d" is added to the nucleotide symbol—dAMP (with a single exception noted below).

Nucleotide Bases

Nomenclature

First of all, we are concerned with the five major bases—adenine, guanine, cytosine, uracil, and thymine—briefly designated by the initial letters of their English names. A, G, C, and U are found in RNA; A, G, C, and_T are found in DNA.

Ribonucleotides are designated as AMP, GMP, CMP, and UMP. Their older, yet still used names are adenylic, guanylic, cytidylic, and uridylic acids, respectively (or adenylate, guanylate, cytidylate, and uridylate for their ionized forms at physiological pH values). Deoxyribonucleotides include dAMP, dGMP, dCMP, and dTMP. The latter is more commonly designated as TMP (or thymidylate), without the prefix d, because T is found exclusively in deoxyribonucleotides (we will not discuss rare exceptions).

If it is necessary to designate a nucleotide without specifying a particular base, the abbreviation NMP or 5'-NMP is often used for ribonucleotides, and dNMP or 5'-dNMP for deoxyribonucleotides.

Deoxy-UMP occurs only as an intermediate in dTMP synthesis; it is not present in DNA (except in cases of chemical damage to the DNA).

The corresponding ribonucleosides (base-sugar) for deoxyribonucleosides are adenosine, guanosine, cytidine, uridine, along with d-adenosine, and so on.

The names cytidine and uridine sound similar to the names of free purine bases (compare adenine and guanine), whereas the name of the free base cytosine resembles the name of the nucleoside (compare with adenosine), so one must be careful here. Transfer RNA also contains "minor" bases (see p. 287). One of these is hypoxanthine. In the biosynthetic pathway of purine bases, hypoxanthine ribotide and inosine monophosphate are also formed; hypoxanthine ribotide is called inosinate.

Structure of the Bases

First of all, it should be noted that A and G belong to Purines, whereas C, U, and T belong to Pyrimidines.

These terms originate from the corresponding chemical structures of purine and pyrimidine, which do not occur in nature in a free state.

Hereafter, these cycles will be depicted in a simplified form.

The structure of the bases composing the nucleotides is shown in Fig. 18.1.

Fig. 18.1. Schematic representation of the Structure of Purine and pyrimidine bases found in nucleic acids

Attachment of bases in nucleotides

Bases are attached to the carbohydrate component of the nucleotide via the nitrogen atom at position 9 of the purine and position 1 of the pyrimidine. The glycosidic bond has the β-configuration; it is located above the plane of the sugar ring. The structures of AMP and CMP are shown in Fig. 18.2.

Fig. 18.2. Structure of purine and pyrimidine nucleotides

Synthesis of purine and pyrimidine nucleotides

Purine nucleotides

Most Cells can synthesize purine bases de novo from lower-molecular-weight precursors. In this process, the bases are not synthesized in a free state; rather, the purine ring is assembled step-by-step by attaching the necessary fragments to ribose-5-phosphate, so that the complete nucleotide is formed by the time ring closure is finished. This applies exclusively to de novo purine synthesis, since free purine bases generated by nucleotide degradation are reused for their resynthesis (see p. 222). The Mechanism of attachment to ribose-5-phosphate (ribosylation) is identical in both cases. The same mechanism is also employed during the synthesis of pyrimidine nucleotides (although the compounds undergoing ribosylation differ). This brings us to PRPP, a metabolite involved in ribosylation.

5-Phosphoribosyl-1-pyrophosphate (PRPP)

PRPP is formed from ribose-5-phosphate—which, in turn, is produced in the Pentose Phosphate Pathway (see Chapter 13)—via The transfer of a pyrophosphate group from ATP catalyzed by the enzyme PRPP synthetase.

PRPP is an "activated" form of ribose-5-phosphate; the respective enzymes can build upon it to form nucleotide bases and release

pyrophosphate. The reaction is thermodynamically driven by the Hydrolysis of the latter to 2P_i.

In this reaction, the configuration of the C-1 carbon atom changes such that the base adopts the required β-position.

Note that in de novo synthesis (see reaction 1 in Fig. 18.4), simply an NH₂ group derived from glutamine acts as the base, yielding 5-phosphoribosylamine as the product. During purine salvage, a preformed purine is used as the base. Typically, the term nucleotide is associated with purine or pyrimidine bases, but it can also apply to other bases linked to sugar phosphate in a similar manner.

Returning from the General Overview to de novo purine synthesis: following The formation of 5-phosphoribosylamine, 9 consecutive reactions take place, culminating in the assembly of the first purine nucleotide with hypoxanthine as its base (Fig. 18.3). This nucleotide is IMP, or inosinic acid.

Fig. 18.3. Schematic representation of the de novo Synthesis of the purine nucleotides GMP, AMP, and XMP. The base in IMP, or inosine monophosphate, is hypoxanthine. The base in XMP, or xanthosine monophosphate, is xanthine

IMP serves as a metabolic crossroad because its base, hypoxanthine, is converted into either adenine or guanine, yielding AMP or GMP, respectively. This pathway is outlined in Fig. 18.3, and all reactions of this process are presented in Figs. 18.4 and 18.5. Let us discuss the most interesting reactions. The synthesis of 1 molecule of a purine nucleotide consumes 6 molecules of ATP. In this process, only phosphate groups are cleaved from ATP, while the adenine nucleotide itself remains unaltered, meaning the overall net result is the synthesis of AMP. Let us examine reactions (3) and (9) in Fig. 18.4, which are of particular importance as they involve the transfer of single-carbon units.

Fig. 18.4. Pathway of de novo purine nucleotide Biosynthesis from PRPP to inosinic acid. Reactions indicated by circled numbers are discussed in the text. Structural changes resulting from each successive step of the cycle are highlighted

Fig. 18.5. Synthesis of GMP and AMP from inosinic acid (IMP). Structural changes resulting from the respective reaction are highlighted

One-carbon transfer reactions in purine nucleotide synthesis

Synthesis reactions (3) and (9) involve the incorporation of a formyl (HCO-) group into biosynthetic intermediates. The donor of these groups in both cases is

N10-formyltetrahydrofolate. Within The Cell, tetrahydrofolate (FH₄) acts as a carrier of formyl groups. It is a coenzyme derived from the vitamin Folic acid (F), also known as pteroylglutamic acid.

Vitamin F is reduced to FH₄ in a two-stage process requiring NADPH.

Note that in the structure of FH₄, the N-5 and N-10 atoms are positioned such that only a single carbon atom can bridge the gap between them. For our purposes, FH₄ can be represented as follows:

And N¹⁰-formyltetrahydrofolate as follows:

This serves as the formyl group donor in reactions (3) and (9) of Purine Biosynthesis; these reactions are catalyzed by formyltransferase.

Where does the formyl group in formyl-FH₄ come from?

The formyl group originates from The amino acid Serine (which is readily synthesized from the glycolytic intermediate 3-phosphoglycerate).

The enzyme serine hydroxymethylase transfers a hydroxymethyl group (-CH₂OH) to FH₄, yielding Glycine and N⁵, N¹⁰-methylene-FH₄.

The resulting methylene-FH₄ is not yet ready to participate in formylation, because the CH₂ group is more reduced than a formyl group.

The methylene group is oxidized by an NADP⁺-dependent enzyme to a methenyl derivative, which upon hydrolysis is converted into N¹⁰-formyl-FH₄, the formyl group donor.

How are ATP and GTP synthesized from AMP and GMP?

Most synthetic reactions within the cell require nucleoside triphosphates, primarily for the synthesis of nucleic acids. A simple yet highly efficient solution exists: the cell utilizes kinase enzymes that carry out high-energy phosphate group transfers between nucleotides. Because the Free energy change in this reaction is negligible, phosphate transfer from one nucleotide to another proceeds readily. ATP serves as the primary source of phosphate groups. It is important to note that the energy generated through metabolic processes is continuously consumed to regenerate ATP from ADP and P_i. De novo synthesized AMP and GMP are subsequently phosphorylated by Kinases, as illustrated below.

Salvage pathway of purine bases in nucleotide synthesis

We have already emphasized that de novo purine synthesis does not yield free purine bases; Purine nucleotides are formed directly. However, there is an alternative pathway for purine nucleotide synthesis in which free bases are converted into nucleotides following interaction with PRPP. These free bases are generated during nucleotide degradation; rather than undergoing further breakdown, they are conserved. Two enzymes—specifically, two phosphoribosyltransferases—participate in this process. One generates nucleotides from adenine, while the other acts on hypoxanthine and guanine. The latter enzyme, known as HGPRT (hypoxanthine-guanine phosphoribosyltransferase), catalyzes the following reaction.

Apparently, adenine salvage plays a less significant role in humans compared to guanine and hypoxanthine, given that the primary degradation pathways of these nucleotides lead to the formation of free hypoxanthine (from AMP) and guanine (from GMP) (Fig. 18.6).

Fig. 18.6. Schematic of the synthesis of free purine bases from hypoxanthine and guanine produced during nucleotide degradation. Patients lacking adenosine deaminase in their lymphocytes suffer from immunodeficiency. The Treatment of this condition marked the first successful application of Gene Therapy—introducing the normal adenosine deaminase gene in vitro into Bone Marrow stem cells, followed by their transplantation back into the patient.

What is The Physiological Role of Purine Base Salvage?

Since purine synthesis demands a substantial amount of energy, the mechanism for salvaging free purine bases is highly advantageous as it allows the cell to limit de novo synthesis. Furthermore, erythrocytes, for instance, are incapable of de novo purine synthesis and rely solely on available preformed bases.

The physiological importance of purine salvage is particularly highlighted by a genetic disorder in children known as Lesch-Nyhan syndrome, which is associated

with a deficiency of the enzyme HGPRT. This condition leads to neurological abnormalities, manifesting as mental retardation, motor coordination deficits, and self-injurious aggressive behavior that often results in various physical traumas. Because de novo synthesis is relatively weak in the Brain, this organ is exceptionally sensitive to disruptions in base Salvage Pathways. The absence of purine salvage reactions in such patients is accompanied by excessive de novo purine nucleotide synthesis in The Liver and elevated levels of PRPP. This explains the dramatically increased production of uric acid—similar to that seen in Gout—which can lead to Kidney Damage caused by urate crystals. The exact link between the biochemical defect and the neurological symptoms in Lesch-Nyhan syndrome remains unclear. Although uric acid overproduction can be mitigated with allopurinol, this treatment does not alleviate the neurological symptoms. Notably, patients with regular gout do not exhibit such neurological manifestations.

There are likely multiple sources of free purine bases utilized for purine resynthesis. Although dietary products contain purines in the form of nucleic acids, evidence suggests that the majority of these are degraded by intestinal epithelial cells and do not enter the bloodstream. Conversely, purines introduced directly into the bloodstream are utilized by cells. The liver serves as the primary site of purine synthesis; research has proven that it releases purines into the Blood for use by other cells, such as reticulocytes, which lack a complete de novo synthetic system. It is also possible that cells utilize purine bases derived from The breakdown of nucleic acids. Lysosomal degradation of cellular components containing nucleic acids presumably liberates primarily free bases. Although The Significance of synthesizing purine bases from molecular fragments is undisputed, a complete understanding of how free bases are transported throughout the body is still lacking.

The reutilization of previously formed purine bases naturally offers a distinct energetic advantage, resulting in a corresponding decrease in their de novo synthesis. This is achieved through two mechanisms: first, salvage lowers PRPP levels and thereby diminishes THE CONTRIBUTION OF the entire pathway to purine production; second, the AMP and GMP generated via purine base salvage exert feedback inhibition.

Formation of uric acid from purines

Nucleotide degradation leads to the formation of free hypoxanthine and guanine. A portion of these bases is reconverted into nucleotides, while the remainder is oxidized to produce uric acid (Fig. 18.7).

Fig. 18.7. Conversion of hypoxanthine and guanine into uric acid. The drug allopurinol is structurally similar to hypoxanthine. Upon the action of xanthine oxidase, allopurinol is converted into alloxanthine, which in turn inhibits xanthine oxidase.

The enzyme xanthine oxidase, responsible for uric acid production, is localized primarily in the liver and intestinal mucosal cells. Gout is caused by elevated blood urate levels, accompanied by the deposition of urate crystals in Tissues. The primary source of uric acid is likely the excessive de novo production of purine nucleotides, triggered in some patients by the heightened activity of PRPP synthetase. An excess of purine nucleotides can also result from HGPRT deficiency. The pharmaceutical drug allopurinol, used in the treatment of gout, shares a structural similarity with hypoxanthine.

Inside the body, allopurinol is converted by xanthine oxidase itself into alloxanthine, an inhibitor of xanthine oxidase. This process is known as suicide inhibition, or self-inactivation. It leads to the preferential accumulation of xanthine and hypoxanthine rather than uric acid (see Fig. 18.7). These intermediates are more Water-soluble than uric acid and are excreted from the body more rapidly, thereby preventing the deposition of insoluble uric acid crystals in tissues and relieving the Clinical symptoms of gout.

Regulation of purine nucleotide synthesis

As with all metabolic pathways, strict regulation of purine nucleotide synthesis is essential to prevent biochemical chaos. The de novo pathway serves as a classic example of allosteric feedback inhibition. The "control point" is the first (reversible) reaction of de novo purine synthesis, which is catalyzed by PRPP synthetase and inhibited by AMP, ADP, GMP, and GDP. The subsequent enzyme catalyzing the first irreversible reaction of purine nucleotide synthesis (see Fig. 18.4, reaction 2) is inhibited in a similar manner (Fig. 18.8).

Fig. 18.8. Simplified diagram illustrating the Regulation of the purine nucleotide biosynthesis pathway. Positive feedback regulation by ATP and GTP occurs at the level of energy supply for the reactions.

However, this is not the end of the story, as de novo synthesis yields IMP, which is then utilized to produce AMP and GMP. This reaction is regulated by end-product feedback inhibition (see Fig. 18.8). Both positive and negative regulatory mechanisms ensure the balanced production of ATP and GTP, since both nucleotides are required for nucleic acid biosynthesis (see Chapter 19).

Synthesis of Pyrimidine Nucleotides

Most body cells synthesize pyrimidine nucleotides de novo. Unlike in Bacteria, the salvage pathway for free pyrimidine bases is not as prominent in mammals as it is for purines. However, the nucleoside thymidine is readily phosphorylated to TMP by thymidine kinase; thus, the resynthesis of this nucleotide does take place.

The pyrimidine synthesis pathway is schematically outlined in Fig. 18.9 and shown in full in Fig. 18.10. It begins with aspartic acid and leads to the formation of a cyclic compound, orotic acid. In a PRPP-dependent reaction, orotic acid is converted into the corresponding nucleotide, which in turn is converted into UMP. Similar to purines, UTP is formed through the action of kinase enzymes, and CTP is produced by the amination of UTP.

Fig. 18.9. The synthesis of pyrimidine nucleotides involves The conversion of CTP to CDP. The complete pyrimidine synthesis pathway is presented in Fig. 18.10

Fig. 18.10. The de novo pathway of pyrimidine nucleotide synthesis. Structural changes that occurred in the preceding reaction are highlighted in color

How Are Deoxyribonucleotides Formed?

DNA Synthesis requires dATP, dGTP, dCTP, and dTTP (see Chapter 19). The reduction of ribonucleotides to their deoxy derivatives occurs at the diphosphate level, using NADPH as the reducing agent (the electron transfer process to the reductase is not shown).

The resulting dADP, dGDP, dCDP, and dUDP are converted into triphosphates via phosphorylation by the respective kinases using ATP. However, dUTP does not participate in DNA synthesis because DNA contains thymine (methylated uracil) as one of its bases, rather than uracil. dUTP is converted into dTTP in a three-step process. First, dUTP is hydrolyzed to dUMP:

dUTP + Н₂O —> dUMP + PP_i.

Next, dUMP is converted into dTMP, which is then phosphorylated to form dTTP. An allosteric feedback regulation system maintains the balanced production of the four deoxynucleotide triphosphates.

Conversion of dUMP to dTMP

The methylation of dUMP is of particular interest. The enzyme involved in this process, thymidylate synthase, utilizes methylene-tetrahydrofolate (N⁵, N¹⁰-methylene FH₄) as a coenzyme.

The scheme below shows only the most relevant part (for the issues discussed) of N⁵, N¹⁰-methylene-FH₄.

Recall that in purine synthesis, the methylene group of FH₄ is oxidized to form a formyl group. During thymidylate synthesis, the methylene group is transferred and simultaneously reduced to the methyl group of thymine; the reaction is catalyzed by thymidylate synthase. FH₄ serves as the reducing agent, being converted into FH₂ in the process.

The FH₂ generated in this reaction is reduced back to FH₄ by the action of Dihydrofolate Reductase. In turn, FH₄ can be converted into methylene-FH₄ via a reaction with serine:

To sustain thymidylate synthesis, FH₂ must be continuously reduced to FH₄ by dihydrofolate reductase. Antileukemic drugs such as methotrexate (amethopterin) and aminopterin (referred to as antifolates), acting as structural analogs of folate, inhibit FH₂ reductase.

The structure of methotrexate (amethopterin) shown below does not need to be memorized; aminopterin has a similar structure, but lacks the N¹⁰-methyl group.

Cancer cells, such as leukemic cells, require the rapid production of dTMP for DNA synthesis, and this process can be selectively inhibited by these drugs, as illustrated in Fig. 18.11.

Fig. 18.11. MECHANISM OF ACTION of the antineoplastic drug methotrexate (schematic). Compare its structure with that of folic acid shown on p. 219

Tetrahydrofolate, Vitamin B₁₂, and Pernicious Anemia

In addition to participating in the reaction above, N⁵, N¹⁰-methylene-FН₄ can be reduced to N⁵-methyl-FН₄, which provides methyl groups for the conversion of homocysteine to Methionine (see p. 199) in a reaction catalyzed by methionine synthase. The latter requires vitamin B₁₂ as a cofactor (see p. 136). In its absence—for instance, in pernicious anemia—the available reserves of FН₄ are converted into methyl-FН₄ and become unavailable for participation in purine nucleotide biosynthesis and other reactions. The "methyl trap" hypothesis explains why vitamin B₁₂ deficiency causes megaloblastic anemia, which is identical to that observed in folate deficiency. The neurological symptoms of pernicious anemia may be caused by methylmalonic acidemia (see p. 136).

Although bacteria possess several vitamin B-dependent reactions, only two are known in humans. These reactions are catalyzed by methylmalonyl-CoA mutase (see p. 136) and methionine synthase. Vitamin B12 deficiency in pernicious anemia is primarily due to the lack of intrinsic factor—a gastric glycoprotein required for intestinal absorption of the vitamin—rather than to inadequate dietary intake.

Questions for Chapter 18

1. Describe the mechanism of ribosylation.

2. What cofactor is involved in two reactions of purine nucleotide synthesis? Provide the structural formulas of its formyl derivative.

3. What is the Origin of the formyl group? Using the structural formulas of the cofactor, explain how it is generated.

4. What is the function of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)?

5. Lesch-Nyhan syndrome encompasses several Genetic Disorders. Explain their biochemical basis.

6. How does the drug allopurinol reduce The production of uric acid?

7. Draw a diagram illustrating the Main Pathways of Allosteric Regulation in purine nucleotide synthesis.

8. Certain compounds in the body are methylated using methionine as a source of methyl groups. Is this true in the case of thymidine monophosphate synthesis? Justify your answer.

9. How does the antileukemic drug methotrexate inhibit cancer cell Replication?

10. The symptoms of pernicious anemia somewhat resemble those associated with folate deficiency. What is the possible reason for this?