BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 10. NUCLEIC ACID METABOLISM

10.5. Nucleotide Biosynthesis

10.5.1. Biosynthesis of Purine Nucleotides

De novo Biosynthesis OF PURINE NUCLEOTIDES. Studies using compounds containing stable isotopes ¹⁴C and ¹⁵N have helped identify the precursors of nucleotides. It was established that The formation of the purine ring involves the Amino Acids aspartate, Glycine, and glutamine, alongside CO₂ and two single-carbon derivatives of tetrahydrofolic acid: methenyl-H₄-folate and formyl-H₄-folate (Fig. 10.7).

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Fig. 10.7. Sources of purine ring atoms

The pathway of purine nucleotide biosynthesis is generally similar in mammals, birds, Yeast, and Bacteria. The process consists of a series of sequential reactions facilitated by specific Enzymes. During the Synthesis of purine nucleotides, a free nitrogenous base is not formed; instead, the purine ring is assembled directly on a ribose-5-phosphate moiety. The first reaction begins with The transfer of the amide group of glutamine to PRPP, yielding 5-phosphoribosyl-1-amine. This reaction is catalyzed by the regulatory enzyme glutamine-PRPP amidotransferase (purine nucleotide synthase). Subsequently, the amino group of 5-phosphoribosyl-1-amine is sequentially joined by a glycine residue, N^5,10-methenyl-H₄-folate, another amide group from glutamine, carbon dioxide, the amino group of aspartic acid, and a formyl residue from N¹⁰-formyl-H₄-folate (Fig. 10.8).

Fig. 10.8. Synthesis of inosine 5'-monophosphate

A characteristic feature of inosine 5'-monophosphate (IMP) synthesis is the involvement of tetrahydrofolate (FH₄) in two separate single-carbon transfer reactions. One utilizes N¹⁰-methenyl-FH₄, while the other employs N¹⁰-formyl-FH₄. The latter is derived from N^5,10-methenyl-FH₄, which in turn is a product of the NADP-dependent dehydrogenation of N^5,10-methylene-FH₄. While N^5,10-methylene-FH₄ serves as a source of one-carbon units for numerous acceptors, N^5,10-methenyl-FH₄ supplies the one-carbon group (either directly or via the formation of N¹⁰-formyl-FH₄) exclusively for purine bases.

The entire ten-step series culminates in the Formation of the first purine nucleotide, IMP, the synthesis of which consumes at least six ATP molecules. IMP serves as a common precursor for the synthesis of adenylic and guanylic acids. Additionally, it is found in small amounts in tRNA as one of the minor nucleotides.

Conversion of IMP into AMP and GMP. AMP and GMP are synthesized from inosinic acid through the Modification of the hypoxanthine ring, with each process occurring in two distinct stages (Fig. 10.9).

Fig. 10.9. Synthesis of AMP and GMP from IMP

The synthesis of AMP is catalyzed by adenylosuccinate synthetase at the expense of GTP energy by replacing the oxygen atom at the C-6 position of the purine ring with an amino group donated by aspartic acid, yielding adenylosuccinate. In the second step, adenylosuccinate lyase cleaves adenylosuccinate, releasing fumarate and forming AMP.

The Synthesis of the second purine nucleotide, GMP, also proceeds in two steps. First, IMP is oxidized by an NAD-dependent IMP dehydrogenase to produce xanthylic acid, followed by the replacement of the oxygen atom at the C-2 position with an amino group provided by the amide group of glutamine. This transamidation is catalyzed by GMP synthetase utilizing ATP energy.

It is important to note that during the formation of purine nucleotides, ATP is expended in the synthesis of GMP, whereas GTP is consumed in the synthesis of AMP. This reciprocal utilization of purine nucleoside phosphates for The production of end products helps maintain an equivalent balance of purine nucleotides within Cells.

For synthesized purine nucleoside monophosphates to participate in the synthesis of Nucleic Acids, certain Coenzymes, and numerous other synthetic and energy-yielding reactions, they must be converted into their active forms. These activated nucleotides are nucleoside 5'-di- and triphosphates.

The formation of phosphorylated purine nucleotides (ADP, GDP, ATP, GTP) from their respective monophosphates (AMP and GMP) occurs through their interaction with ATP, mediated by base-specific nucleoside monophosphate Kinases. For instance, adenylate kinase catalyzes the reaction:

The ADP thus formed is subsequently phosphorylated and converted into ATP either via substrate-level phosphorylation during Glycolysis or through Oxidative Phosphorylation in the Respiratory Chain.

Phosphorylation of GMP occurs analogously; however, unlike AMP—which yields two molecules of ADP—the reaction produces GDP and ADP:

In the next step, GDP is phosphorylated through the action of ATP and the enzyme nucleoside diphosphate kinase:

Synthesis of purine nucleotides via Salvage Pathways. The de novo pathway of purine nucleotide synthesis cannot fully cover the body's demand for these compounds. This high requirement for purine nucleotides led to The Development of "salvage" pathways of synthesis.

The sources of purine bases for this synthesis are purine bases and nucleosides. Their salvage occurs via two pathways:

1. PRPP-dependent phosphoribosylation of purine bases. This mechanism is catalyzed by two enzymes. Specifically, adenine reacts with PRPP in the presence of adenine phosphoribosyltransferase to form AMP:

Guanine salvage proceeds similarly, but is mediated by a different enzyme—hypoxanthine-guanine phosphoribosyltransferase:

This same enzyme participates in the salvage of hypoxanthine (formed via adenine deamination), converting it into inosinic acid (IMP):

This method of purine salvage is the most efficient, as the IMP produced by hypoxanthine-guanine phosphoribosyltransferase is directly channeled into AMP and GMP synthesis.

2. Phosphorylation of purine ribonucleosides. Nucleosides generated during the Catabolism of nucleic acids from nucleotides by nucleotidases can undergo re-phosphorylation to form nucleoside-5'-monophosphates through the transfer of a γ-phosphate group from ATP to the corresponding substrate. This phosphorylation reaction is catalyzed by adenosine kinase:

In addition, adenosine kinase phosphorylates 2'-deoxyadenosine and also exhibits some activity toward guanosine, inosine, and their 2'-deoxy derivatives.

Regulation of purine nucleotide synthesis. The synthesis of a single IMP molecule requires the Hydrolysis energy of six ATP molecules, utilizing glycine, glutamine, tetrahydrofolic acid precursors, and aspartate as building blocks. To conserve energy and nutritional resources, efficient Regulation of the de novo purine nucleotide biosynthesis pathway is essential.

The key compound governing purine nucleotide synthesis is PRPP. The intracellular concentration of PRPP depends on the rates of its synthesis, utilization, and degradation. The level of PRPP is determined by two factors: the availability of synthesis substrates—primarily ribose-5-phosphate—and The activity of PRPP synthetase, which is sensitive to inorganic phosphate and purine nucleotide concentrations. The enzyme is activated by phosphate and inhibited by purine nucleoside mono-, di-, and triphosphates, whose inhibitory potency follows the order: NMP > > NDP > NTP (Fig. 10.10). The activity of glutamine-PRPP amidotransferase is inhibited via negative feedback by the End products of the reaction chain—IMP, AMP, and GMP.

Fig. 10.10. Regulation of purine nucleotide synthesis:

1 - PRPP synthetase; 2 - glutamine-PRPP amidotransferase; 3 - adenylosuccinate synthetase; 4 - IMP dehydrogenase;

5 - adenylosuccinate lyase; 6 - GMP synthetase. O indicates feedback inhibition by end products

The de novo synthesis of AMP and GMP is also regulated at their branch points: AMP inhibits the activity of adenylosuccinate synthetase, while GMP inhibits the formation of xanthylate catalyzed by IMP dehydrogenase. Thus, through this mechanism, an excess of either AMP or GMP suppresses its own synthesis from IMP without affecting the synthesis of the other nucleotide. However, as noted above, Cross-Regulation of the divergent pathways of IMP METABOLISM also exists: the formation of adenylosuccinate from IMP on the path to AMP is stimulated by GTP, whereas the formation of GMP from xanthylate requires the presence of ATP. Such reciprocal regulation prevents The biosynthesis of one purine nucleotide when the other is in short supply.