Nucleotides Metabolism and De Novo Synthesis of Nucleotides

Let us make an in-depth study of the nucleotides metabolism with special emphasis on de novo synthesis of nucleotides.

Nucleotide consists of a purine or pyrimidine base plus a pentose sugar (ribose or deoxyribose) and a phosphoryl group (H₃PO₄). The purine ring consists of a 5-membered imidazol ring fused to a six-membered ring structure with two common or bridge carbon atoms (C-4 and C-5) and contains 4-N atoms. The pyrimidine ring has a simpler struc­ture with only a six-membered ring with two N-atoms.

The metabolism of nucleotides includes synthesis, inter-conversions, and catabolism of various purine and pyrimidine nucleotides which are schematically shown in Fig. 9.32 and 9.33 respectively. Metabolism of nucleotides is clearly known in animals than in plant cells.

Purine and pyrimidine nucleotides can be synthesized in living organisms either by (i) de novo pathways, or (ii) salvage pathways. In de novo pathways, the synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose-5-phosphate, CO₂, and NH₃. In salvage pathways, the breakdown products of nucleotides i.e. free bases and nucleosides are salvaged and recycled back to synthesize nucleotides again.

Salvage pathways may involve reconstruction of nucleotides from free bases by addition of ribose-phosphate moiety, or by phos­phorylation of nucleosides. De novo pathways are more important quantitatively than salvage path­ways. However, by using salvage pathways for nucleotides synthesis, the cells do conserve energy.

De Novo Synthesis of Purine Nucleotides (IMP, AMP & GMP):

i. Free bases are not intermediates in de novo pathways of nucleotides synthesis i.e., they are not synthesized and then attached to ribose phosphate.

ii. The purine ring structure is built up one or a few atoms at a time, and is gradually attached to ribose phosphate throughout the process.

iii. Phosphoribosyl pyrophosphate (PRPP), is an important intermediate and the starting point in purine nucleotide synthesis. It is formed from α-D-Ribose-5-phosphate (Fig. 9.34)

iv. (The ultimate precursors of the purine ring are shown in Fig. 9.35. These precursors were established from information obtained from isotopic experiments with ¹⁴C or ¹⁵N- labeled precursors that were administered into pigeons and tracing the incorporation of labeled atoms into the purine ring of their excreted uric acid).

iv. IMP (Inosine monophosphate or Inosinate) is the first purine nucleotide to be synthesized.

v. IMP is then converted into AMP and GMP.

Formation of IMP from PRPP:

a. Synthesis of IMP from PRPP (phosphoribosyl pyrophosphate) takes place in ten differ­ent steps as shown in Fig. 9.36.

b. The 5-membered imidazol ring is added first to PRPP; the remaining six-membered ring of purine is built up afterwards.

(i) In the first committed step of this pathway, phosphoribosylamine is formed by the action of the enzyme glutamine phosphoribosyl pyrophosphate amidotransferase. An amino group supplied by glutamine is attached to C-1 of PRPP and there is inversion of configuration at C-1, from a to p position. The purine ring is subsequently built on this structure. Atom no. 9 (N-9) of the purine ring is introduced in this first step.

(ii) In the second step, the enzyme synthetase forms an amide bond between carboxyl group of glycine and amino group of phosphoribosylamine forming 5′-phosphoribosyl glycinamide. ATP is hydrolyzed to provide energy. The atoms 4, 5 and 7 of the purine ring are introduced in this step.

(iii) In the third step, C-8 of the purine ring is introduced as a formyl group donated by 10-formyl tetrahydrofolate, in presence of the enzyme formyl transferase, so that 5′- phosphoribosyl-N-formyl glycinamide is formed.

(iv) In the fourth step, N-3 of purine ring is introduced by transfer of another amino group from glutamine to phosphoribosyl formyl glycinamide by a synthetase enzyme, form­ing 5′-phosphoribosyl-N-formyl glycinamidine. ATP is hydrolyzed and provides en­ergy.

(v) In the fifth step, cyclization reaction occurs in presence of synthetase, Mg²⁺, and K⁺ ions, so that imidazol ring is closed. The product is 5′-phosphoribosyl-5- aminoimidazole.

(vi) In the sixth step, C-6 of the purine ring is introduced by addition of bicarbonate (CO₂ + H₂O → HCO₃^–) in presence of a specific carboxylase enzyme. The product of this reaction is 5′-phosphoribosyl-5-aminoimidazole-4-carboxylate.

(vii) In the seventh step, N-8 of the purine ring is contributed by aspartate. The latter forms an amide with 4-carboxyl group in presence of synthetase, and a succinocarboxamide is formed. ATP is hydrolyzed and provides energy.

(viii) 5′-phosphoribosyl – 4 – (N-succino carboxamide) – 5-aminoimidazole is now cleaved in presence of adenylosuccinate lyase to release formate and forming 5′-phosphoribosyl- 4-carboxamide – 5 – aminoimidazole.

(ix) In the ninth step, the final atom of purine ring (i.e., C-2) is introduced which is supplied by a formyl group from 10-formyl tetrahydrofolate to the 5-amino group of the almost completed ribonucleotide.

(x) In the last step, a second ring closure takes place by elimination of water to form IMP. The enzyme involved is IMP-cyclohydrolase (IMP-synthase)

Synthesis of IMP from ribose-5-phosphate requires a total of six high energy phos­phate groups from ATPs (assuming hydrolysis of pyrophosphate (P-P)) released in step (i).

Conversion of IMP into AMP and GMP:

a. IMP is converted into AMP (adenosine mono-phosphate) and GMP (guanosine monophosphate) by two different pathways, each consisting of two steps.

(i) Conversion of IMP into AMP:

(a) In the first step of this pathway, keto group of IMP is first displaced by the amino of aspartate to produce adenylosuccinate in the presence of the enzyme synthetase. GTP is hydrolyzed and provides energy.

(b) Adenylosuccinate is now cleaved non-hydrolytically by the enzyme adenylosuccinate lyase to produce fumarate and the purine nucleotide AMP (adenosine mono-phosphate.)

(ii) Conversion of IMP into GMP:

(a) In the first step of this pathway, there is dehydrogenation of IMP to xanthosine- 5-phosphate (XMP), in the presence of NAD⁺-dependent IMP-dehydrogenase.

(b) The second step involves transfer of an amino group from glutamine to C-2 of the xanthine ring to produce GMP (guanosine monophosphate). ATP is hydrolyzed to provide energy, while glutamate is released.

b. (After the formation of purine mononucleotides, purine di and tri-nucleotides may be synthe­sized by addition of one or two more phosphoryl groups respectively).

De Novo synthesis of Pyrimidine Nucleotides:

I. Common pyrimidine ribonucleotides are cytidine-5′-monophosphate (CMP or cytidylate) and uridine-5′-monophosphate (UMP or uridylate).

II. De novo biosynthesis of pyrimidine nucleotides is simpler than those of purine nucleotides because of the simpler structure of pyrimidine ring.

III. In contrast to the de novo biosynthetic pathway of purine nucleotides, in pyrimidine biosynthetic pathway the pyrimidine ring is constructed before ribose – 5 – phosphate is incorporated into the nucleotide.

IV. Orotidine-5′-monophosphate (OMP), is the first pyrimidine nucleotide to be synthesized. From OMP, pathways lead to synthesis of nucleotides of uracil, cytosine and thymine.

De novo synthesis of pyrimidine nucleotides is illustrated in Fig. 9.37, a brief description of this follows:

(i) The first step in this pathway is the synthesis of carbamoyl phosphate from CO₂ and NH₄⁺ by carbamoyl phosphate from CO₂ and NH₄⁺ by carbamoyl phosphate synthetase. NH₄⁺ is supplied by glutamine.

(ii) In the next step, carbamoyl phosphate reacts with aspartate to form carbamoyl aspartate. The reaction is catalyzed by aspartate carbamoyl transferase.

(iii) In the third step, the pyrimidine ring is closed by dehydroorotase to form dihydroorotate with elimination of water molecule.

(iv) Dihydroorotate is now oxidized to orotate by dehydrogenase enzyme.

(v) In the fifth step, orotate is converted into orotidine-5′-monophosphate (OMP) by the enzyme orotate phosphoribosyl transferase. The ribose phosphate moiety is supplied by PRPP (phosphoribosyl pyrophosphate).

(vi) In the last reaction, OMP is decarboxylated by OMP-decarboxylase to yield UMP (uridine monophosphate).

(v) UMP is the precursor of other pyrimidine nucleotides. First, UMP is phosphorylated to UTP (uridine triphosphate). Then, CTP (cytidine triphosphate) is formed from UTP by the action of the enzyme cytidylate synthetase. ATP is hydrolyzed and provides energy; NH₄⁺ is supplied by glutamine (Gln) which is converted into glutamate (Glu). See Fig. 9.37.

Formation of Deoxy Ribonucleotides:

a. Deoxy nucleotides are formed by reduction of corresponding ribonucleotides by ribonucleotide reductases, 2′-OH group of the ribopentose sugar is replaced with hy­drogen.

b. Thymidylate (TMP) is formed from dUMP

TMP is synthesized in the cells from dUMP (deoxy uridine monophosphate) and the latter can be formed by two different pathways:

(i) Mainly, by deamination of dCMP (deoxycytidine-monophosphate) in the presence of the enzyme deoxy-cytidylate deaminase.

(ii) Also, by reduction of UDP to dUDP followed by phosphorylation of dUDP to dUTP. The latter is then hydrolyzed to dUMP.

c. dUMP is now methylated to from thymidylate (TMP) by the enzyme thymidylate synthase. The methyl group is provided by 5, 10-methylene tetrahydrofolate.

Catabolism (Degradation) Of Nucleotides:

(1) Catabolism of Purine Nucleotides:

The Pathway of degradation of purine nucleotides is illustrated in Fig. 9.38. In primates, birds, and some other animals, the end product of this pathway is uric acid.

a. Uric acid is further catabolized to other excretory products in different groups of other animals (Fig. 9.39). Most mammals other than primates oxidize uric acid further to allantoin. In many other animals, allantoin is catabolized further to allantoic acid (as in bony fishes), urea (as in amphibians and cartilaginous fishes), or ammonia and CO₂ (as in marine invertebrates).

b. No ATPs are formed during catabolism of purine nucleotides.

(2) Catabolism of Pyrimidine Nucleotides:

I. Ribose phosphate is released during catabolism prior to destruction of base.

II. Pyrimidines are catabolized to β-alanine, NH₃, and CO₂.

III. Thymine is catabolized to β-aminoisobutyrate.

IV. As in case of catabolism of purine nucleotides, no ATPs are formed in pyrimidine nucleotides catabolism.