BIOSYNTHESIS OF PURINE
1. INTRODUCTION
Nucleotides are essential for many cellular functions, including
the storage of genetic information, gene expression, energy
metabolism, cell signaling, and biosynthesis. In a cell,
nucleotides exist primarily as 5'-triphosphates. ATP is the most
prevalent nucleotide, reaching mM concentrations in many cell
types, while other nucleotides may be present at much lower
concentrations (cAMP).
nucleobase
Adenine
Guanine
Thymine
Cytosine
Uracil
Hypoxanthine
Xanthine
Nucleoside
Adenosine
Guanosine
Thymidine
Cytidine
Uridine
Inosine
Xanthosine
Nucleotide 5’-monophosphate
Adenosine
5’-monophosphate
(adenylate, AMP)
Guanosine
5’-monophosphate
(guanylate, GMP)
Thymidine
5’-monophosphate
(thymidylate, TMP)
Cytidine
5’-monophosphate
(cytidylate, CMP)
Uridine
5’-monophosphate
(uridylate, UMP)
Inosine
5’-monophosphate
(inosinate, IMP)
Xanthosine
5’-monophosphate
(xanthylate , XMP)
1
Purine Biosynthesis
DE NOVO SYNTHESIS OF PURINES
. IMP is synthesized first and serves as a precursor to adenine and
guanine nucleotides.
Reaction scheme:
2
Step 1: Formation of 5-phosphoribosyl-1-amine from PRPP
(committed step) by the action of glutamine phosphoribosyl
amidotransferase. PRPP provides the foundation for purine
biosynthesis. Glutamine phosphoribosyl amidotransferase has two
domains: one hydrolyzes glutamine to ammonia, and the other one
is phosphoribosyl transferase domain. Ammonia is channeled to
site II without leaving the enzyme.
2-
Future N-9 of the purine ring
Glu + NH3
Gln + H2O
2-
O3PO
O3PO
CH2
CH2
O
-O
O
-O
P
O
OH
NH2
O
-configuration of
the sugar
OP
O
PPi is replaced by NH2
O inversion of configuration
OH
PPi
OH
OH
5-Phosphoribosyl-1-amine
5-phosphoribosyl-1-pyrophosphate (PRPP)
This is the committed step. It is feedback inhibited by IMP. GMP,
AMP
Step 2: Glycine is
phosphoribosylamine
coupled
to
the
amino
group
of
glycinamide ribonucleotide synthetase
ATP + Gly
N7
ADP + Pi
C4 C5
NH
P-ribose
P-ribose-NH2
phorphoribosyl
amine
Gly is coupled to the
amino group
-
O
C
CH2
C
O
NH3+
NH3+
CH2
This is the only step
in purine
biosynthesis that
contributes more
than one atom to the
purine skeleton.
glycinamide
ribonucleotide
O
a) The carboxylate group of Gly is activated by phosphorylation to
form
acylphosphate.
O
-O
P O
O-
NH3+
C
O
CH2
3
b) The phosphate is then displaced by the amino group of
phophoribosylamine.
O
O
-O
P O
-O
NH3+
C
OP-ribose-NH2
P OH
O- P-ribose-NH
CH2
NH3+
C
O
CH2
O
Step 3: Transfer of a formyl group from N10formyltetrahydrofolate to the amino group of the glycine residue
(catalyzed by glycinamide ribonucleotide transferase). N10-formyltetrahydrofolate (THF) serves as a C1 carbon donor.
O
C8
10
N -formylNH3+
P-ribose
NH
C
THF
H
NH N7
THF
CH2
O
C
NH
P-ribose
C4
C
H2 N
N
OH
O
Formylglycinamide ribonucleotide
Glycinamide ribonucleotide
CH2
5
N
H
CH2 C5
N9
 -amino terminus
is formylated
H
N
N
O
C
H
CH2
O
N
C
O-
O
10
C
N
H
H2 H2 O
CH C C C OH
H
10
N -formyl -tetrahydrofolate (THF)
Step 4: The inner amide group is converted to an amidine by the
replacement with ammonia derived from glutamine:
O
O
H
P-ribose
NH
C
ATP
H
ADP + Pi
NH
C
C
C8
NH N7
N9
CH2
P-ribose
Glu + NH3
Gln + H2O
O
Formylglycinamide ribonucleotide
NH
N3
=NH replaces =O
4
C4
C
CH2 C5
N
H
Formylglycinamidine ribonucleotide
Note: Steps 1-4 are catalyzed by a multienzyme complex. Many of
the intermediates in purine biosynthesis are unstable in aqueous
solution and only exist in solvent protected environment. The
formation of multienzyme complexes makes it possible to
internally channel the product of one reaction to the next catalytic
center without solvent exposure.
Step 5: An intramolecular coupling reaction accompanied by a loss
of water forms the five-membered imidazole ring. The carbonyl is
O
AIR synthetase
C
H
ATP
NH
N7
N
C8
ADP + Pi
HC
CH C5
NH
P-ribose
C
CH2
P-ribose
- H2O
N
N
N9
C C4
N3 NH2
ring closure
H
Formylglycinamidine ribonucleotide
5-aminoimidazole ribonucleotide
activated by phosphorylation and the phosphate is displaced by an
amino group as shown on p. 5 (top)
Step 6: Bicarbonate adds first to the exocyclic amino group and
then is transferred to the neighboring carbon atom of the imidazole
ring:
ATP +
O
HO
N
N
HC
N
O-
CH
HC
CH
N
C
P-ribose
ADP + Pi
- H2O
NH2
8
carboxyl transfer HC
O
C
9N
N
7
O
5
C
6
O-
C 4
HN
P-ribose
carboxylation of the
amino group
O-
P-ribose
NH2
Carboxyaminoimidazole
ribonucleotide
5-aminoimidazole ribonucleotide
ATP +
O
HO
H
N
Enzyme:
AIR carboxylase
HC
CH
O-
H
N
ADP + Pi
HC
N
CH
C
8
carboxyl transfer HC
H 7
N
O
5
C
6
O
O-
C
9N
C 4
- H Ocarboxylate is phosphorylated,
Step 7:N The
imidazole
and the
HN
P-ribose
P-ribose
NH
P-ribose
NH
phosphate is displaced by the amino group ofO- aspartate:
2
2
2
carboxylation of the
amino group
Carboxyaminoimidazole
ribonucleotide
5-aminoimidazole ribonucleotide
5
ATP
O
N
ADP + Pi
HC
O-
C
P-ribose
- H2O
H2N
Carboxyaminoimidazole
ribonucleotide
CH CO2-
CH
CH2
NH2
CO2-
5-aminoimidazole-4-(N-succinylcarboxamide)
ribonucleotide
CH2
Asp
N
H
C
N
P-ribose
NH2
CO2-
C
C
N
O
N
HC
CO2-
Enzyme: SAICAR synthetase
Step 8. Elimination of fumarate is catalyzed by lyase. The carbon
skeleton of aspartate is lost as fumarate (only the amino group is
contributed by aspartate).
CO2CH
fumarate
HC
HC
N
CO2-
O
N
C
N
H
C
P-ribose
CH
N1
HC
CH
H
NH2
O
N
CO2-
N
CO2-
C
NH2
C
P-ribose
NH2
5-aminoimidazole-4-carboxamide ribonucleotide
5-amidoimidazole-4-(N-succinylcarboxxamide)
ribonucleotide
Step 9: The second formyl group is added from N10formyltetrahydrofolate:
O
N
HC
N
P-ribose
10
N -formylTHF
C
O
N
HC
THF
C
NH2
C
N
NH2
P-ribose
5-aminoimidazole-4-carboxamide
ribonucleotide (AICAR)
NH2
C
NH
C2
C
H
O
5-formaminoimidazole-4-carboxamide
ribonucleotide (FAICAR)
6
Enzyme: AICAR transformylase
Step 10: Cyclization/dehydration produces inosinate:
HC
N
7
O
N
8
NH2
C
P-ribose
NH
9
C
N
4
P-ribose
H
C
O
5
HC
AMP cyclohydrolase
C
N
6
C
3
NH 1
N
H2O
CH
2
O
Inosinate (IMP)
5-formaminoimidazole-4-carboxamide
ribonucleotide (FAICAR)
Formation of AMP and GMP from IMP
IMP is a common precursor to both AMP and GMP.
1. Formation of adenylate (AMP): the C-6 carbonyl group of
inosinate is replaced with the amino group from Asp.
O-
Adenylsuccinate synthetase
O
IMP dehydrogenase
O
O
GDP + Pi
H2O
N
NH
N
- H2O
C
ATP
N
N
NN
H
P-ribose
NN
P-ribose
P-ribose
P-ribose
H2N
(IMP)
(IMP)
Asp =
CH C
CH2
C
O-
Xanthylate
Adenylsuccinate
(XMP)
H
H
O
O
COO-
AMP + PPi
NH2
N N
NH
O- H2O
O
O
Inosinate
Inosinate
CH
HN
NH
NH
N
N
N
NADH + H+
N
N
fumarate
CH
NAD+
Asp
H
GMP synthetase
GTP
O
-OOC
C
N
N
N
H3N
P-ribose
P-ribose
+
lyaseH2O + Gln
Glu
N
N
Guanylate (GMP)
Adenylate (AMP)
O
OH
Note that GTP is used as a phosphate donor
2. Formation
of
guanylate
(GMP).
Inosinate(Inosine
instead of ATP
monophosphate) is first oxidized to xanthylate, and the C-2
carbonyl is then converted to an amino group.
7
NH2
Regulation of Purine Biosynthesis:
 AMP, GMP and IMP are feedback inhibitors of purine
biosynthesis
 Purine biosynthesis is energetically expensive (6 moles ATP
used per 1 mol IMP)
 Nucleotide concentrations are tightly regulated in a cell. Loss
of regulation may lead to clinical disease (e.g. gout).
1. PRPP synthesis is inhibited by AMP, IMP, GMP (indicators
of poor energy status)
2. The committed step (phosphoribosylamine formation) is
inhibited by all purine mononucleotides.
3. AMP and GMP inhibit their own synthesis from IMP
nucleotide
Reciprocal substrate regulation balances the relative purine
levels:
AMP synthesis requires GTP as the energy source
GMP synthesis requires ATP as the energy source
Purine biosynthesis:
1. Purines are synthesized using both de novo and salvage
pathways.
2. The purine ring is built on a ribose skeleton to make IMP,
followed by branches to AMP and GMP. Amino acids serve as N
8
donors, while CO2 and C1 units of N10-formyl-THF are carbon
donors.
3.
The
general
mechanism
involves
ATP-mediated
phosphorylation of carbonyl oxygen, followed by phosphate
displacement by an amine.
4.
Purine biosynthesis is tightly regulated via feedback
inhibition. Formation of phosphoribosylamine is the committed
step. The loss of regulation can lead to a clinical disease.
5.
NAD+ is biosynthesized from nicotinate, ATP, and PRPP.
Glutamine is used as an amino donor.
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