Synthesis of Purine Nucleotides

SYNTHESIS OF PURINE NUCLEOTIDES

The atoms of the purine ring are contributed by a number of compounds, including amino acids (aspartate, glycine, and glutamine), CO₂, and N¹⁰-formyltetrahydrofolate (Figure 22.5). The purine ring is constructed primarily in the liver by a series of reactions that add the donated carbons and nitrogens to a preformed ribose 5-phosphate.

Figure 22.5 Sources of the individual atoms in the purine ring. The order in which the atoms are added is shown by the numbers in the black boxes (see Figure 22.7).

A. Synthesis of 5-phosphoribosyl-1-pyrophosphate

5-Phosphoribosyl-1-pyrophosphate (PRPP) is an “activated pentose” that participates in the synthesis and salvage of purines and pyrimidines. Synthesis of PRPP from ATP and ribose 5-phosphate is catalyzed by PRPP synthetase (Figure 22.6). This X-linked enzyme is activated by inorganic phosphate and inhibited by purine nucleotides (end-product inhibition). [Note: The sugar moiety of PRPP is ribose, and, therefore, ribonucleotides are the end products of de novo purine synthesis. When deoxyribonucleotides are required for DNA synthesis, the ribose sugar moiety is reduced.]

Figure 22.6 Synthesis of PRPP, showing the activator and inhibitors of the reaction. [Note: This is not the committed step of purine synthesis because PRPP is used in other pathways.] P= phosphate; Pi = inorganic phosphate; AMP = adenosine monophosphate.

B. Synthesis of 5-phosphoribosylamine

Synthesis of 5-phosphoribosylamine from PRPP and glutamine is shown in Figure 22.7. The amide group of glutamine replaces the pyrophosphate group attached to carbon 1 of PRPP. This is the committed step in purine nucleotide biosynthesis. The enzyme, glu-tamine:phosphoribosylpyrophosphate amidotransferase, is inhibited by the purine 5 -nucleotides AMP and guanosine monophosphate ([GMP] also called guanylate), the end products of the pathway. The rate of the reaction is also controlled by the intracellular concentration of PRPP. [Note: The concentration of PRPP is normally far below the Michaelis constant (K_m) for the amidotransferase. Therefore, any small change in the PRPP concentration causes a proportional change in rate of the reaction.]

Figure 22.7 De novo synthesis of purine nucleotides, showing the inhibitory effect of some structural analogs. AMP = adenosine monophosphate; ADP = adenosine diphosphate; GMP = guanosine monophosphate; PRPP = 5-phosphoribosyl- 1-pyrophosphate; Pi = inorganic phosphate; PPi = pyrophosphate.

C. Synthesis of inosine monophosphate, the “parent” purine nucleotide

The next nine steps in purine nucleotide biosynthesis leading to the synthesis of inosine monophosphate ([IMP] whose base is hypoxanthine) are illustrated in Figure 22.7. Four steps in this pathway require ATP as an energy source, and two steps in the pathway require N¹⁰-formyltetrahydrofolate as a one-carbon donor. [Note: Hypoxanthine is found in tRNA.]

D. Synthetic inhibitors of purine synthesis

Some synthetic inhibitors of purine synthesis (for example, the sulfonamides), are designed to inhibit the growth of rapidly dividing microorganisms without interfering with human cell functions (see Figure 22.7). Other purine synthesis inhibitors, such as structural analogs of folic acid (for example, methotrexate), are used pharmacologically to control the spread of cancer by interfering with the synthesis of nucleotides and, therefore, of DNA and RNA (see Figure 22.7).

Inhibitors of human purine synthesis are extremely toxic to tissues, especially to developing structures such as in a fetus, or to cell types that normally replicate rapidly, including those of bone marrow, skin, gastrointestinal (GI) tract, immune system, or hair follicles. As a result, individuals taking such anticancer drugs can experience adverse effects, including anemia, scaly skin, GI tract disturbance, immunodeficiencies, and hair loss.

E. Synthesis of adenosine and guanosine monophosphate

The conversion of IMP to either AMP or GMP uses a two-step, energy-requiring pathway (Figure 22.8). Note that the synthesis of AMP requires guanosine triphosphate (GTP) as an energy source, whereas the synthesis of GMP requires ATP. Also, the first reaction in each pathway is inhibited by the end product of that pathway. This provides a mechanism for diverting IMP to the synthesis of the purine present in lesser amounts. If both AMP and GMP are present in adequate amounts, the de novo pathway of purine synthesis is turned off at the amidotransferase step.

Figure 22.8 Conversion of IMP to AMP and GMP showing feedback inhibition. [Note: AMP is also called adenylate. GMP is also called guanylate.] NAD(H) = nicotinamide adenine dinucleotide; GDP = guanosine diphosphate; GTP = guanosine triphosphate; AMP = adenosine monophosphate; Pi = inorganic phosphate; PPi = pyrophosphate.

F. Conversion of nucleoside monophosphates to nucleoside diphosphates and triphosphates

Nucleoside diphosphates are synthesized from the corresponding nucleoside monophosphates by base-specific nucleoside monophosphate kinases (Figure 22.9). [Note: These kinases do not discriminate between ribose or deoxyribose in the substrate.] ATP is generally the source of the transferred phosphate because it is present in higher concentrations than the other nucleoside triphosphates. Adenylate kinase is particularly active in the liver and in muscle, where the turnover of energy from ATP is high. Its function is to maintain equilibrium among the adenine nucleotides (AMP, ADP, and ATP). Nucleoside diphosphates and triphosphates are interconverted by nucleoside diphosphate kinase, an enzyme that, unlike the monophosphate kinases, has broad substrate specificity.

Figure 22.9 Conversion of nucleoside monophosphates to nucleoside diphosphates and triphosphates.

AMP = adenosine monophosphate;

ADP = adenosine diphosphate;

GMP = guanosine monophosphate;

GDP = guanosine diphosphate;

GTP = guanosine triphosphate;

CDP = cytidine diphosphate;

CTP = cytidine triphosphate.

G. Salvage pathway for purines

Purines that result from the normal turnover of cellular nucleic acids, or the small amount that is obtained from the diet and not degraded, can be converted to nucleoside triphosphates and used by the body. This is referred to as the “salvage pathway” for purines. [Note: Salvage is particularly important in the brain.]

1. Salvage of purine bases to nucleotides: Two enzymes are involved: adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Both enzymes use PRPP as the source of the ribose 5-phosphate group (Figure 22.10). The release of pyrophosphate and its subsequent hydrolysis by pyrophosphatase makes these reactions irreversible. [Note: Adenosine is the only purine nucleoside to be salvaged. It is phosphorylated to AMP by adenosine kinase.]

Figure 22.10 Salvage pathways of purine nucleotide synthesis. [Note: Virtually complete deficiency of HGPRT results in Lesch-Nyhan syndrome. Partial deficiencies of HGPRT are known. As the amount of functional enzyme increases, the severity of the symptoms decreases.]

IMP = inosine monophosphate;

GMP = guanosine monophosphate;

AMP = adenosine monophosphate;

PRPP = 5-phosphoribosyl-1- pyrophosphate;

PPi = pyrophosphate.

2. Lesch-Nyhan syndrome: Lesch-Nyhan is a rare, X-linked recessive disorder associated with a virtually complete deficiency of HGPRT. The deficiency results in an inability to salvage hypoxanthine or guanine, from which excessive amounts of uric acid, the end product of purine degradation, are then produced. In addition, the lack of this salvage pathway causes increased PRPP levels and decreased IMP and GMP levels. As a result, glutamine:phosphoribosylpyrophosphate amidotransferase (the regulated step in purine synthesis) has excess substrate and decreased inhibitors available, and de novo purine synthesis is increased. The combination of decreased purine reutilization and increased purine synthesis results in increased degradation of purines and the production of large amounts of uric acid, making Lesch-Nyhan a heritable cause of hyperuricemia. In patients with Lesch-Nyhan syndrome, the hyperuricemia frequently results in the formation of uric acid stones in the kidneys (urolithiasis) and the deposition of urate crystals in the joints (gouty arthritis) and soft tissues. In addition, the syndrome is characterized by motor dysfunction, cognitive deficits, and behavioral disturbances that include self-mutilation (for example, biting of lips and fingers) as shown in Figure 22.11).

Figure 22.11 Lesions on the lips of Lesch-Nyhan patients caused by self-mutilation.