Purine Bases Can Be Synthesized de Novo or Recycled by Salvage Pathways

for the hydrolysis of the side chain carboxamide of glutamine to generate ammonia. Key residues in this active site
include a cysteine residue and a histidine residue.
III. Synthesizing the Molecules of Life
25. Nucleotide Biosynthesis
25.1. In de Novo Synthesis, the Pyrimidine Ring Is Assembled from Bicarbonate, Aspartate, and Glutamine
Figure 25.5. Substrate Channeling. The three active sites of carbamoyl phosphate synthetase are linked by a channel
(yellow) through which intermediates pass. Glutamine enters one active site, and carbamoyl phosphate, which includes
the nitrogen atom from the glutamine side chain, leaves another 80 Å away.
III. Synthesizing the Molecules of Life
25. Nucleotide Biosynthesis
25.2. Purine Bases Can Be Synthesized de Novo or Recycled by Salvage Pathways
Purine nucleotides can be synthesized in two distinct pathways. First, purines are synthesized de novo, beginning with
simple starting materials such as amino acids and bicarbonate (Figure 25.6). Unlike the case for pyrimidines, the purine
bases are assembled already attached to the ribose ring. Alternatively, purine bases, released by the hydrolytic
degradation of nucleic acids and nucleotides, can be salvaged and recycled. Purine salvage pathways are especially noted
for the energy that they save and the remarkable effects of their absence (Section 25.6.2).
25.2.1. Salvage Pathways Economize Intracellular Energy Expenditure
Free purine bases, derived from the turnover of nucleotides or from the diet, can be attached to PRPP to form purine
nucleoside monophosphates, in a reaction analogous to the formation of orotidylate. Two salvage enzymes with different
specificities recover purine bases. Adenine phosphoribosyltransferase catalyzes the formation of adenylate
whereas hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyzes the formation of guanylate as well as
inosinate (inosine monophosphate, IMP), a precursor of guanylate and adenylate (Section 25.2.4).
Similar salvage pathways exist for pyrimidines. Pyrimidine phosphoribosyltransferase will reconnect uracil, but not
cytosine, to PRPP.
25.2.2. The Purine Ring System Is Assembled on Ribose Phosphate
De novo purine biosynthesis, like pyrimidine biosynthesis, requires PRPP, but for purines, PRPP provides the foundation
on which the bases are constructed step by step. The initial committed step is the displacement of pyrophosphate by
ammonia, rather than by a preassembled base, to produce 5-phosphoribosyl-1-amine, with the amine in the β
configuration.
Glutamine phosphoribosyl amidotransferase catalyzes this reaction. This enzyme comprises two domains: the first is
homologous to the phosphoribosyltransferases in salvage pathways, whereas the second produces ammonia from
glutamine by hydrolysis. However, this glutamine-hydrolysis domain is distinct from the domain that performs the same
function in carbamoyl phosphate synthetase. In glutamine phosphoribosyl amidotransferase, a cysteine residue located at
the amino terminus facilitates glutamine hydrolysis. To prevent wasteful hydrolysis of either substrate, the
amidotransferase assumes the active configuration only on binding of both PRPP and glutamine. As is the case with
carbamoyl phosphate synthetase, the ammonia generated at the glutamine-hydrolysis active site passes through a channel
to reach PRPP without being released into solution.
25.2.3. The Purine Ring Is Assembled by Successive Steps of Activation by
Phosphorylation Followed by Displacement
Nine additional steps are required to assemble the purine ring. Remarkably, the first six steps are analogous
reactions. Most of these steps are catalyzed by enzymes with ATP-grasp domains that are homologous to those in
carbamoyl phosphate synthetase. Each step consists of the activation of a carbon-bound oxygen atom (typically a
carbonyl oxygen atom) by phosphorylation, followed by the displacement of a phosphoryl group by ammonia or an
amine group acting as a nucleophile (Nu).
De novo purine biosynthesis proceeds as follows (Figure 25.7).
1. The carboxylate group of a glycine residue is activated by phosphorylation and then coupled to the amino group of
phosphoribosylamine. A new amide bond is formed while the amino group of glycine is free to act as a nucleophile in
the next step.
2. Formate is activated and then added to this amino group to form formylglycinamide ribonucleotide. In some
organisms, two distinct enzymes can catalyze this step. One enzyme transfers the formyl group from N 10formyltetrahydrofolate (Section 24.2.6). The other enzyme activates formate as formyl phosphate, which is added
directly to the glycine amino group.
3. The inner amide group is activated and then converted into an amidine by the addition of ammonia derived from
glutamine.
4. The product of this reaction, formylglycinamidine ribonucleotide, cyclizes to form the five-membered imidazole ring
found in purines. Although this cyclization is likely to be favorable thermodynamically, a molecule of ATP is consumed
to ensure irreversibility. The familiar pattern is repeated: a phosphoryl group from the ATP molecule activates the
carbonyl group and is displaced by the nitrogen atom attached to the ribose molecule. Cyclization is thus an
intramolecular reaction in which the nucleophile and phosphate-activated carbon atom are present within the same
molecule.
5. Bicarbonate is activated by phosphorylation and then attacked by the exocyclic amino group. The product of the
reaction in step 5 rearranges to transfer the carboxylate group to the imidazole ring. Interestingly, mammals do not
require ATP for this step; bicarbonate apparently attaches directly to the exocyclic amino group and is then transferred to
the imidazole ring.
6. The imidazole carboxylate group is phosphorylated again and the phosphate group is displaced by the amino group of
aspartate. Thus, a six-step process links glycine, formate, ammonia, bicarbonate, and aspartate to form an intermediate
that contains all but two of the atoms necessary for the formation of the purine ring.
Three more steps complete the ring construction (Figure 25.8). Fumarate, an intermediate in the citric acid cycle, is
eliminated, leaving the nitrogen atom from aspartate joined to the imidazole ring. The use of aspartate as an amino-group
donor and the concomitant release of fumarate are reminiscent of the conversion of citrulline into arginine in the urea
cycle and these steps are catalyzed by homologous enzymes in the two pathways (Section 23.4.2). A formyl group from
N 10-formyltetrahydrofolate is added to this nitrogen atom to form a final intermediate that cyclizes with the loss of
water to form inosinate.
25.2.4. AMP and GMP Are Formed from IMP
A few steps convert inosinate into either AMP or GMP (Figure 25.9). Adenylate is synthesized from inosinate by the
substitution of an amino group for the carbonyl oxygen atom at C-6. Again, the addition of aspartate followed by the
elimination of fumarate contributes the amino group. GTP, rather than ATP, is the phosphoryl-group donor in the
synthesis of the adenylosuccinate intermediate from inosinate and aspartate. In accord with the use of GTP, the enzyme
that promotes this conversion, adenylsuccinate synthase, is structurally related to the G-protein family and does not
contain an ATP-grasp domain. The same enzyme catalyzes the removal of fumarate from adenylosuccinate in the
synthesis of adenylate and from 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide in the synthesis of inosinate.
Guanylate (GMP) is synthesized by the oxidation of inosinate to xanthylate (XMP), followed by the incorporation of an
amino group at C-2. NAD+ is the hydrogen acceptor in the oxidation of inosinate. Xanthylate is activated by the transfer
of an AMP group (rather than a phosphoryl group) from ATP to the oxygen atom in the newly formed carbonyl group.
Ammonia, generated by the hydrolysis of glutamine, then displaces the AMP group to form guanylate, in a reaction
catalyzed by GMP synthetase. Note that the synthesis of adenylate requires GTP, whereas the synthesis of guanylate
requires ATP. This reciprocal use of nucleotides by the pathways creates an important regulatory opportunity (Section
25.4).
III. Synthesizing the Molecules of Life
25. Nucleotide Biosynthesis
25.2. Purine Bases Can Be Synthesized de Novo or Recycled by Salvage Pathways
Figure 25.6. de Novo Pathway for Purine Nucleotide Synthesis. The origins of the atoms in the purine ring are
indicated.
III. Synthesizing the Molecules of Life
25. Nucleotide Biosynthesis
25.2. Purine Bases Can Be Synthesized de Novo or Recycled by Salvage Pathways
Figure 25.7. de Novo Purine Biosynthesis. 1. Glycine is coupled to the amino group of phosphoribosylamine. 2. N 10Formyltetrahydrofolate transfers a formyl group to the amino group of the glycine residue. 3. The inner amide group is
phosphorylated and converted into an amidine by the addition of ammonia derived from glutamine. 4. An intramolecular
coupling reaction forms the five-membered imidazole ring. 5. Bicarbonate adds first to the exocyclic amino group and
then to a carbon atom of the imidazole ring. 6. The imidazole carboxylate is phosphorylated, and the phosphate is
displaced by the amino group of aspartate.
III. Synthesizing the Molecules of Life
25. Nucleotide Biosynthesis
25.2. Purine Bases Can Be Synthesized de Novo or Recycled by Salvage Pathways
Figure 25.8. Inosinate Formation. The removal of fumarate, the addition of a second formyl group from N 10formyltetrahydrofolate, and cyclization completes the synthesis of inosinate (IMP), a purine nucleotide.
III. Synthesizing the Molecules of Life
25. Nucleotide Biosynthesis
25.2. Purine Bases Can Be Synthesized de Novo or Recycled by Salvage Pathways
Figure 25.9. Generating AMP and GMP. Inosinate is the precursor of AMP and GMP. AMP is formed by the addition
of aspartate followed by the release of fumarate. GMP is generated by the addition of water, dehydrogenation by NAD+,
and the replacement of the carbonyl oxygen atom by -NH2 derived by the hydrolysis of glutamine.