Biochemistry of Medicinals II (Phar 6152)

Spring 2004
Instructor: Dr. Natalia Tretyakova, Ph.D.

BIOSYNTHESIS OF NUCLEOTIDES
Required reading: Stryer’s Biochemistry 5th edition Chapter 25 (p. 693-712) and 262-268
(or Stryer’s Biochemistry 4th edition Chapter 29 (p. 739-759)

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).

Biological significance of nucleotides

1. Building blocks of nucleic acids (DNA and RNA).

2. Involved in energy storage, muscle contraction, active transport, and maintenance of
ion gradients.
3. Activated intermediates in biosynthesis (e.g. UDP-glucose, S-adenosylmethionine
(SAM).
4. Components of coenzymes (NAD+, NADP+, FAD, FMN, and CoA)
5. Metabolic regulators:
a. Second messengers (cAMP, cGMP)
b. Phosphate (PO32- ) donors for phosphorylation of kinases and phosphatases in
signal transduction (ATP)
c. Regulation of some enzymes via adenylation and uridylylation

Each nucleotide contains a purine or pyrimidine base, a ribose or deoxyribose sugar, and
a phosphate:
Nucleotide

Purine or
Pyrimidine
O Base
5'
-O P O
O
H H 1'
O- 4'
H 3' 2' H
O H(OH)
Phosphate
Pentose sugar
Nucleoside

1
-glycosidic bond between the C-1’ of the
sugar and the N9 of purine or N1 of
pyrimidine

2. NOMENCLATURE OF NUCLEIC BASES, NUCLEOSIDES, AND

NUCLEOTIDES

NH 2 O O O

7 6
N N N
N 5 1 N
NH NH NH
N N
8

4 2 N N N
N NH2 N
9N N N H
N H N O
H H H H
3
Purine Guanine (G) Xanthine
Adenine (A) Hypoxanthine

NH2 O
O
4
5 3 H3C
N N NH
NH

6 2
N1 N O N O
H N O
H H H

Pyrimidine Cytosine (C) Thymine (T) Uracil (U)

nucleobase nucleoside Nucleotide 5’-monophosphate

Adenine Adenosine Adenosine 5’-monophosphate (adenylate, AMP)
Guanine Guanosine Guanosine 5’-monophosphate (guanylate, GMP)
Thymine Thymidine Thymidine 5’-monophosphate (thymidylate, TMP)
Cytosine Cytidine Cytidine 5’-monophosphate (cytidylate, CMP)
Uracil Uridine Uridine 5’-monophosphate (uridylate, UMP)
Hypoxanthine Inosine Inosine 5’-monophosphate (inosinate, IMP)
Xanthine Xanthosine Xanthosine 5’-monophosphate (xanthylate , XMP)

Sources of nucleotides in a cell:

1) Degradation of nucleic acids (salvage
pathways). Free purine bases can be
recycled by coupling with the ribose
phosphate moiety, 5-phospho-ribosyl-1-
pyrophosphate (PRPP), to form nucleotide
monophosphates:

2
 adenine phosphoribosyl
transferase
Adenine + PRPP Adenylate + PPi
Hypoxanthine-guanine phosphoribosyl
transferase (HGPRT)
Guanine + PRPP Guanylate + PPi

Hypoxanthine + PRPP Inosinate+ PPi

Ribose phosphate

2-
O3PO
Biodegradation ofCHPurines
2
O -O
O -O
O-
Nucleotides undergo continual Pturnover
P in a cell. Nucleic acids are degraded to
O O O
nucleotides by the action
OH of nucleases. Nucleotides are hydrolyzed to nucleosides by
OH

nucleotidases. Nucleosides
PRPP are degraded to free bases and ribose or ribose-1-phosphate
by nucleoside phosphatases: Salvage Degradation
2-
O3P HO
O Base Base
O Nucleootidase O Base
nucleoside phosphorylase
H H H H
H H H H HO
OH OH OH OH
O
H H
Nucleotide 5'-phosphate Nucleoside
2-
H OPO3
OH OH
Ribose-1-phosphate

phosphoribo
2- mutase
O3PO
OH
O
H H
PRPP H
OH OH

Ribose-5-phosphate
The end product of purine degradation in humans is uric acid. Adenosine deaminase
converts adenosine to inosine, which is hydrolyzed to its free base, hypoxanthine, by the
action of purine nucleoside phosphorylase. Hypoxanthine and xanthine are substrates for
xanthine oxidase, which ultimately converts purines to uric acid:

3
Pathological conditions resulting from defects in nucleotide metabolism:
1. Gout (excess accumulation of uric acid due to
defects in uric acid excretion or HGPRT deficiency
).
Allopurinol inhibits xanthine oxidase, preventing the
accumulation of uric acid.
2. Lesch-Nyhan syndrome (HGPRT null)
3.
Immunodeficiency (deficiency of adenosine deaminase
).
2) De novo synthesis of nuclotides

2. DE NOVO SYNTHESIS OF PYRIMIDINES

Metabolic precursors of nucleotides include amino acids, CO2, and ribose-5-phosphate.

Sources of carbon and nitrogen atoms in pyrimidine ring

O O

from carbamoyl phosphate Xanthine oxidase

NH NH
C N N
4
N3 5C N N
from aspartate
H
N O H
N
H
O

2 6C Allopurinol
C 1
Alloxanthine
NH2 C
N -O
CH2

O O
CH
PO3- H2N
COO-
2-
O3PO
CH2 O -O
O -O O- 5-phosphoribosyl-1-pyro-
P P phosphate (PRPP) is the
O O O source of phosphoribose
OH OH

The pyrimidine ring is synthesized in a 6-step process that requires participation of

several enzymes. Most required enzymes are cytosolic, with the exception of orotate
dehydrogenase that is localized in mitochondria (see below). The general strategy is to
use pre-assembled components (carbamoyl phosphate and aspartate) to make a
pyrimidine ring which is then attached to the phosphoribose.

Part 1. The formation of carbamoyl phosphate is catalyzed by cytosolic carbamoyl

phosphate synthetase II (CPS).

(from Gln)
ATP ADP O
O NH3 Pi
O HO
HO HO C
C C O
CPS CPS NH2
O O P
phosphorylation O- ammonia displaces
of bicarbonate the phosphate
O-
Bicarbonate Carboxyphosphate Carbamic acid

4
 O-
ATP ADP O
O -O
O
HO P C Regulated Step
C
O in Pyrimidine
CPS Biosynthesis
NH2
NH2
phosphorylation

Carbamoyl phosphate
Carbamic acid
General Mechanism for
replacing carbonyl oxygen with amino group:
ATP O- NH3 O- Pi R
R R ADP R R

OH NH2
O O P O O P O

R' R' R' NH2 R'

R' O- O-

We will see this mechanism used over and over again in nucleotide biosynthesis.

The ammonia necessary for the formation of carbamic acid originates from glutamine:
NH2 NH2

H 2N H2 CPS HO H2
C CH OH C CH OH
NH3
C C C C C C
H2 H2
O O O O

Glutamine (Gln) glutamate

Note that a single enzyme, carbamoyl phosphate synthetase II, catalyzes all 4 reactions
required for the synthesis of carbamoyl phosphate. Reaction intermediates are channeled
internally between the active sites responsible for phosphorylation, carbamic acid
formation, and glutamine hydrolysis, without dissociating from the enzyme. This is
necessary to protect unstable intermediates from hydrolysis and to drive the reactions
forward. (See Stryer's Biochemistry 5th ed. Fig. 23.3 and 23.4).

Part 2. The formation of orotate

5
 The committed step in the biosynthesis of pyrimidines is the formation of N-carbamoyl-
aspartate from aspartate and carbamoyl phosphate. Carbamoyl aspartate is then cyclized
and oxidized by NAD+ to orotate:
Dihydroorotate
Aspartate O O Dehydrogenase O
Dihydroorotase
Transcarbamoylase
Asp C H+ H2O C NAD+ NADH + H+ C
Pi
NH2 HN NH
O- HN HN NH
O
-O O
P C CH COO CH C C C
OOC OOC OOC
O Asp replaces Pi C cyclization C O C O
NH2 H2 H2 H
oxidation
Carbamoyl phosphate
N-Carbamoylaspartate Dihydroorotate Orotate

NH2 Electrons are

channeled directly
to the transport
CH COO-
-OOC chain of
C mitochondria
H2
Aspartate

Note:
1. Aspartate transcarbamoylase is allosterically inhibited by CTP, the final product of
pyrimidine nucleotide biosynthesis. This ensures a tight control of CTP concentrations in
a cell. See Stryer 5th Ed. p. 262-268 for enzyme structure/further details.
2. N-(phosphonacetyl)-L-aspartate (PALA) is a
O
O
CH2 O-
bisubstrate analog inhibitor of aspartate
C P transcarbamoylase. It resembles
HN O- carbamoylphosphate+ aspartate
-O C C
H2
CH 3. Human carbamoyl phosphate synthetase and
O C O PALA
aspartate transcarbamoylase are part of the same
protein.
O-

Part 3: Formation of UMP. Orotate coupling to ribose in the form of 5-phosphoribosyl-

1-pyrophosphate (PRPP) produces orotidylate (OMP):
O

O C
HN
-
O3PO
pyrimidine phosphoribosyltransferase
C CH
-
CH2 PPi O3PO C
HN NH O -O O
O -O O-
N C
CH2
C P P O COO
OOC C
C O O O O
H OH OH

Orotate 5-phosphoribosyl-1-pyrophosphate (PRPP) OH OH

Orotidylate

PRPP is produced by phosphorylation of ribose-5-phosphate (from pentose phosphate

pathway):

6
 2-
O3PO ATP AMP 2-
O3PO
CH2 CH2 O -O
O O -O O-

P P
PRPP synthetase
O O O
OH
OH OH OH OH

ribose-5-phosphate 5-phosphoribosyl-1-pyrophosphate (PRPP)

Decarboxylation of orotidylate yields uridylate (UMP), a major pyrimidine nucleotide.

O O

C C
HN HN
CH
-
O3PO C H+ CO2 -
CH
O O3PO C
O
N C C
CH2 CH2 N
O COO O H

UMP
synthase
OH OH OH OH

Orotidylate Uridylate (UMP)

Phosphorylation of UMP by kinases gives rise to UDP and UTP:

UMP kinase
UMP + ATP UDP + ADP
nucleotide diphosphate kinase
UDP + ATP UTP + ADP

Formation of cytosine nucleotides

Cytidine triphosphate is formed from uridine triphosphate by the replacement of a
carbonyl group with an amino group. This is accomplished by a mechanism similar to
carbamoyl phosphate synthesis (p. 4). The O4 of the uridine is phosphorylated, followed
by displacement with ammonia. CTP synthetase is feedback-inhibited by CTP.
Gln + H2O NH2
O
Glu C
N
C
HN CH
NH3 4-
O3PO3PO3PO-H2C O C
CH
4-
O3PO3PO3PO-H2C O C C
CH2 N
C O H
CH2 N
O H
ATP ADP + Pi
OH OH
OH OH
CTP
UTP
Feedback Inhibition in Bacteria Pyrimidine Biosynthesis
Nucleotide concentrations and their biosynthesis are highly regulated. This tight control
is achieved by allosteric modulation (nucleotide end products act as negative effectors).

7
 Glutamine + HCO3- + ATP
Examples of feedback inhibition:
PRPP Carbamoyl phosphate synthetase is feedback –
Carbamoyl phosphate inhibited by CTP (see above).

Aspartate transcarbamoylase (ATCase) is inhibited by

CTP.
OMP
Furthermore, the concentrations of key enzymes are
transcriptionally regulated during the cell cycle, with
UMP increased rates of nucleotide synthesis in the late
G1/early S phase of the cell cycle preceding DNA
UDP replication.

UTP

CTP

Orotic acidurea: a rare disease associated with defects in pyrimidine biosynthesis

– Symptoms: anemia, growth retardation, orotic acid excretion in urine.
– Causes: a defect in phosphoribosyl transferase or orotidylate decarboxylase
–
Treatment: patients are fed uridine: U  UMP  UDP  UTP
UTP inhibits CPS II (regulated step), preventing the biosynthesis of orotic acid
Pyrimidine biosynthesis: Take home message
1. Pyrimidines are synthesized using both de novo and salvage pathways.
2. The pyrimidine ring is synthesized from pre-assembled ingredients (carbamoyl
phosphate and aspartate) and then attached to ribose.
3. Pyrimidine biosynthesis is tightly regulated via feedback inhibition (CTP synthetase,
ATCase) and transcriptional regulation (ATCase).
4. Most of the necessary enzymes are located in the cytosol with the exception of
dihydroorotate (localized in the mitochondria).
5. The mammalian enzymes are multifunctional, e.g. CAD (three activities), UMP
synthetase (two activities).

8
3. DE NOVO SYNTHESIS OF PURINES
Sources of carbon and nitrogen atoms in purine ring:

Gly (2)
CO2 (5)

Aspartate (6) 6
C 7
amino 5 N
1N C 8
C N10-THF (3) (the order of incorporation)
2C C
N
N 9
4
3
N10-THF (7) Glutamine
Glutamine amide (1)
amide (4)

General features:
1. Unlike pyrimidines that do not acquire the ribose until the base moiety is synthesized,
purine ring is assembled on PRPP.
2. IMP is synthesized first and serves as a precursor to adenine and guanine nucleotides.
Reaction scheme:

9
 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.

Future N-9 of the purine ring

Gln + H2O Glu + NH3 2-
O3PO
2-
O3PO CH2 NH2
CH2 O -O O
O -O O- -configuration of
P P PPi is replaced by NH2 the sugar
O O O inversion of configuration
OH OH
OH OH PPi

5-phosphoribosyl-1-pyrophosphate (PRPP) 5-Phosphoribosyl-1-amine

This is the committed step. It is feedback inhibited by IMP. GMP, AMP

Step 2: Glycine is coupled to the amino group of phosphoribosylamine

glycinamide ribonucleotide synthetase

This is the only step
ATP + Gly ADP + Pi N7
NH3+
C4 C5
in purine
a) biosynthesis that
NH CH2 contributes more
P-ribose-NH2 P-ribose C
than one atom to the
phorphoribosyl
amine Gly is coupled to the O purine skeleton.
amino group
- glycinamide
O NH3+ ribonucleotide The carboxylate group of
C CH2 Gly is activated by
phosphorylation to form
O
acylphosphate.
O
-O P O NH3+
C CH2
O-
O

b) The phosphate is then displaced by the amino group of phosphoribosylamine.

O
O -O P OH
-O P O NH3+ O- P-ribo s e -NH NH3+
C CH2
O- C CH2

P-ribo s e -NH2 O
O

10
 Step 3: Transfer of a formyl group from N10-formyltetrahydrofolate to the amino group of
the glycine residue (catalyzed by glycinamide ribonucleotide transferase). N10-formyl-
tetrahydrofolate (THF) serves as a C1 carbon donor.

O
C8
10 H
N -formyl- C
H H2N N N
NH3+ THF THF NH N7 CH2
N 5 H O O-
C4
NH CH2 NH CH2 C5 N CH 2 O C
P-ribose C P-ribose C H H 2 H2 O
N9 OH N C N CH C C C OH
 -amino terminus O 10
O is formylated O C H

Glycinamide ribonucleotide Formylglycinamide ribonucleotide H

N10-formyl -tetrahydrofolate (THF)

Step 4: The inner amide group is converted to an amidine by the replacement with
ammonia derived from glutamine:

O
O
C8
C
C ATP ADP + Pi H
H NH N7
NH
N9
C4
NH CH2 C5
NH CH2 P-ribose C
P-ribose C
Glu + NH3 Gln + H2O
N
O N3 H
=NH replaces =O Formylglycinamidine ribonucleotide
Formylglycinamide 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 activated by phosphorylation and the
phosphate is displaced by an amino group as shown on p. 5 (top)
O
AIR synthetase N7
C8
C N
H ATP ADP + Pi HC
NH
CH C5
NH CH2 N
P-ribose C P-ribose N9 C C4
- H2O
N N3 NH2
ring closure
H
Formylglycinamidine ribonucleotide 5-aminoimidazole ribonucleotide

11
Step 6: Bicarbonate adds first to the exocyclic amino group and then is transferred to the
neighboring carbon atom of the imidazole ring:

ATP +
HO O
N 7
N ADP + Pi 8 N O
HC
HC O- CH carboxyl transfer HC 5
CH 6
C
N C O O-
N C 9N C 4
- H2 O
P-ribose HN
P-ribose NH2 P-ribose NH2
carboxylation of the
amino group O-
5-aminoimidazole ribonucleotide Carboxyaminoimidazole
ribonucleotide
ATP +
Enzyme: AIR carboxylaseHO O H
H N H 7
N 8 O
Step 7: The
HC imidazole carboxylate
O- isADP + Pi
phosphorylated,
HC and carboxyl
CH the phosphate
transfer HC
isNdisplaced
5 by
6
the amino group CH
of aspartate: C
N C O O-
N C 9N C 4
- H2O
P-ribose HN
P-ribose NH2 P-ribose NH2
carboxylation of the O
O ATP
amino group ADP + Pi N O-
N
5-aminoimidazole ribonucleotide HC Carboxyaminoimidazole
HC C CO2-
ribonucleotide
C
N CH
O- - H2O N C
N C H CH2
P-ribose CO2-
P-ribose NH2 NH2
H2N CH CO2-
Carboxyaminoimidazole 5-aminoimidazole-4-(N-succinylcarboxamide)
ribonucleotide Asp CH2 ribonucleotide

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).

CO2-
CH fumarate

HC
O CO2-
N N O
HC CO2- HC N1
C C
N CH NH2
N C H CH N C
CO2-
P-ribose NH2 H P-ribose NH2

5-amidoimidazole-4-(N-succinylcarboxxamide) 5-aminoimidazole-4-carboxamide ribonucleotide

ribonucleotide

Enzyme: adenylsuccinate lyase

12
Step 9: The second formyl group is added from N10-formyltetrahydrofolate:

N O
N10-formyl- N O
HC THF HC
C THF
C
NH2
N C NH2
N C
P-ribose NH2 P-ribose NH H
C
5-aminoimidazole-4-carboxamide
C2
ribonucleotide (AICAR) O
5-formaminoimidazole-4-carboxamide
ribonucleotide (FAICAR)

Enzyme: AICAR transformylase

Step 10: Cyclization/dehydration produces inosinate:

7
N O 8 N
5 O
HC HC
C AMP cyclohydrolase C 6
NH2
N C 9 C NH 1
N
4
P-ribose NH H P-ribose N CH
C 3 2
H 2O
O
5-formaminoimidazole-4-carboxamide Inosinate (IMP)
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- H
O -OOC
Adenylsuccinate synthetase C
fumarate
GTP CH H
CH
O COO-
Asp H
GDP + Pi N NH2
N C
O N
NH O-
N N
N - H2O N N
- H2O
N P-ribose N
P-ribose N
P-ribose O lyase
H2N CH C O-
Inosinate Adenylsuccinate
CH2 Adenylate (AMP)
Asp =
(IMP)
C O
OH

Note that GTP is used as a phosphate donor

instead of ATP

13
4. Formation of guanylate (GMP). Inosinate is first oxidized to xanthylate, and the C-
2 carbonyl is then converted to an amino group:
IMP dehydrogenase
GMP synthetase

NAD+ O O
O
H2O
NADH + H+ N AMP + PPi N
N ATP
NH NH
NH
N N
N N N
N H O H3N NH2
H2O + Gln P-ribose
P-ribose
P-ribose +
Glu
Xanthylate Guanylate (GMP)
Inosinate
(XMP)
(IMP)

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

Biosynthesis of NAD+
Nicotinamide adenine dinucleotide (NAD+) and its phosphorylated analog, NADP+, are
important coenzymes that participate in a number of biological processes involving
electron transfer. NAD+ contains an AMP moiety as part of the molecule:

14
 NAD+ is a two electron acceptor (H-)
O H
O O
H H
NH2 Nicotinamide NH2
NH2
O
P N N
-O P O N
O
NH2 R
R

OH OH N
NAD+ NADH
O
N Adenine
N N

-O P O
P O
O Ribose
OH OH

NAD+
NAD+ synthesis requires nicotinate (vitamin B6), which is derived from tryptophan. In the
first step, nicotinate ribonucleotide is formed from nicotinate and PRPP:
COO-

PRPP PPi
COO-
2- N
O3PO
O
N H H Ribose-P
H
H H
Nicotinate OH OH
Nicotinate ribonucleotide

In the following steps, an AMP moiety is transferred from ATP to nicotinate

ribonucleotide to form desamido-NAD+. Finally, the carboxyl group of desamido-NAD is
converted to amide using glutamine as an ammonia donor:

NADP is obtained by phosphorylation of the 2'-OH of the adenine ribose by ATP in the
presence of NAD+ kinase.

15
Purine biosynthesis: Take home message
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 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.

16