BIOCHEMISTRY - L. Stryer - 1984

VOLUME 2

PART III. BIOSYNTHESIS OF MACROMOLECULAR PRECURSORS

CHAPTER 22. BIOSYNTHESIS OF NUCLEOTIDES

22.3. Phosphoribosyl Pyrophosphate is the Donor of the Ribose Phosphate Moiety of Nucleotides

The pathway of Purine Biosynthesis was elucidated in the 1950s through the work of John Buchanan, G. Robert Greenberg, and others. The ribose phosphate moiety of purine and pyrimidine NUCLEOTIDES is derived from 5-phosphoribosyl-1-pyrophosphate (PRPP), a key intermediate in The biosynthesis of Histidine and Tryptophan. PRPP is synthesized from ATP and ribose 5-phosphate, which in turn is formed in the Reactions of the Pentose Phosphate Pathway (Section 15.1). The pyrophosphate group is transferred from ATP to C-1 of ribose 5-phosphate. PRPP is in the α-configuration.

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Fig. 22.5. Space-filling model of 5-phosphoribosyl-1-pyrophosphate (PRPP), the activated sugar donor in nucleotide biosynthesis

22.4. The Purine Ring is Attached to Ribose Phosphate During Its Assembly

The first committed step in de novo purine nucleotide synthesis is The formation of 5-phosphoribosylamine from PRPP and glutamine. The side-chain amino group of glutamine displaces the pyrophosphate group attached to C-1 of PRPP. During this reaction, the α-configuration of the C-1 atom is inverted to the β-configuration. The resulting C—N glycosidic bond has the β-configuration characteristic of natural nucleotides. The driving force for this reaction is the Hydrolysis of pyrophosphate.

The product of The addition of Glycine to phosphoribosylamine is glycinamide ribonucleotide (Fig. 22.6). The Formation of the amide bond between the carboxyl group of glycine and the amino group of phosphoribosylamine consumes one molecule of ATP. The α-amino group of the glycine residue is then formylated by methenyltetrahydrofolate to yield α-N-formylglycinamide ribonucleotide.

Fig. 22.6. The First stage of purine biosynthesis: formation of 5-aminoimidazole ribonucleotide from PRPP. The Essence of these reactions is the Displacement of the pyrophosphate group by the side-chain amino group of glutamine (1), the addition of glycine (2), formylation by methenyltetrahydrofolate (3), transfer of a nitrogen atom from glutamine (4), and dehydration to close the ring (5)

The amide group of this compound is converted into an amidine group. The nitrogen atom is derived from the side chain of glutamine; this reaction consumes one molecule of ATP. Formylglycinamidine ribonucleotide then undergoes a ring-closure reaction to form 5-aminoimidazole ribonucleotide. This intermediate contains the completed five-membered ring of the purine core.

Now begins the next stage of purine Skeleton synthesis—the assembly of the six-membered ring (Fig. 22.7). Three of the six atoms of this ring are already present in aminoimidazole ribonucleotide. The other three atoms are derived from CO₂, aspartate, and formyltetrahydrofolate. The next carbon atom of the six-membered ring is introduced by the carboxylation of aminoimidazole ribonucleotide, yielding 5-aminoimidazole-4-carboxylic acid ribonucleotide.

Fig. 22.7. The Second Stage of purine biosynthesis: formation of inosinate from 5-aminoimidazole ribonucleotide. The essence of these reactions is carboxylation (6), addition of aspartate (7), Cleavage of fumarate (leaving behind the amino group of aspartate) (5), formylation using N¹⁰-formyltetrahydrofolate (9), and dehydration to close the ring (10)

The amino group of aspartate then reacts with the carboxyl group of this intermediate to form 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide. The formation of this amide bond consumes a molecule of ATP. In the subsequent reaction, the carbon Skeleton of the aspartate residue is cleaved off as fumarate, yielding 5-aminoimidazole-4-carboxamide ribonucleotide. It is worth noting that these two reactions effectively convert a carboxylate into an amide. Thus, only the nitrogen atom of aspartate is incorporated into the purine ring. The final atom of the purine ring is derived from N¹⁰-formyltetrahydrofolate. This yields 5-formamidoimidazole-4-carboxamide ribonucleotide, which undergoes dehydration and ring closure to produce inosinate (IMP), containing the complete purine skeleton. The purine base of inosinate is called hypoxanthine.

22.5. AMP and GMP are Formed from IMP

Inosinate (inosine monophosphate, IMP) is the precursor of AMP and GMP (Fig. 22.8). Adenylate is synthesized from inosinate by replacing the carbonyl oxygen at the C-6 position with an amino group. The amino group is donated by aspartate through the addition of this amino acid to the ribonucleotide, followed by the elimination of fumarate. GTP is the donor of the high-energy phosphate bond in the synthesis of adenylosuccinate from inosinate and aspartate. The cleavage of fumarate from both adenylosuccinate and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide is catalyzed by the same enzyme.

Fig. 22.8. AMP and GMP are synthesized from IMP

Guanylate (guanosine monophosphate, GMP) is synthesized by The oxidation of inosinate followed by the insertion of an amino group at the C-2 position. NAD⁺ is the hydrogen acceptor in the oxidation of inosinate to xanthylate (xanthosine monophosphate, XMP). The side-chain amino group of glutamine is then transferred to xanthylate. This reaction consumes two high-energy bonds, as ATP is cleaved to AMP and PPi, which is subsequently hydrolyzed.

In The conversion of inosinate to adenylate and guanylate, the carbonyl oxygen atom is replaced by an amino group. A similar substitution occurs during the synthesis of formylglycinamide ribonucleotide from its corresponding amide precursor (reaction 4 in Fig. 22.6), in the formation of CTP from UTP (Section 22.13), and in the conversion of citrulline to Arginine in The Urea Cycle (Section 18.5). The general principle of these reactions is that the carbonyl oxygen is converted into a derivative that can readily undergo substitution by an amino group. The tautomeric form of the carbonyl group reacts with ATP (or GTP) to form a phosphate ester, which is then attacked by a nucleophilic amine (Fig. 22.9). Inorganic phosphate is then eliminated from the tetrahedral intermediate, completing the reaction. The attacking amine can be NH₃, the amide group of the glutamine side chain, or the α-amino group of aspartate. An intermediate step in reactions of this type involves the release of phosphate, pyrophosphate, or an AMP moiety. For example, in the synthesis of 5-phosphoribosyl-1-amine from PRPP, the amino group of glutamine displaces pyrophosphate (Section 22.4).

Fig. 22.9. Mechanism of the substitution of a carbonyl oxygen by an amino group