Nucleotide Biosynthesis

Berg, Tymoczko and Stryer

7th Edition
Chapter 25, pages 735-753
Probems: none

dihydrofolate reductase
gene amplification
Functions of Nucleotides

1. Activated precursors of RNA and DNA.

2. Activated precursors of many other biosynthetic reactions.

UDP-glucose in glycogen synthesis
CDP-diacylglycerol in phosphoglyceride synthesis

3. ATP universal energy currency.

4. GTP powers movement of macromolecules and ribosomes.

5. Precursors of the cofactors, NAD, FAD and CoA.

6. Metabolic regulation
cAMP
ATP, a substrate for phosphorylation and adenylation.
GTP involved in signaling cascades
Nomenclature
Ribonucleoside = base + ribose
Deoxyribonucleoside = base + deoxyribose
Nucleotide (mono-, di-, tri-) = ribonucleoside + phosphate
de novo Pyrimidine Biosynthesis
Pyrimidine biosynthesis occurs in six
steps.
1. Carbamoyl phosphate synthetase 1. CPSase
( CPSase) catalyzes the first step in
mammalian cells. This is not really
part of the pathway in bacteria since
there is only one CPSase that 2. ATCase
provides carbamoyl phosphate for 3. DHOase
both pyrimdines and arginine.
2. The second and third steps are
catalyzed by ATCase and DHOase
and form the ring.
3. In mammals, the first three
enzymes are fused (CAD).
4. In the last three steps, a double UMP
bond is introduced into the ring,
PRPP is added and the ring is
decaboxylated to form UMP.
 Carbamoyl Phosphate Synthetase

Carbamoyl phosphate synthesis involves four partial reactions.

1. Glutamine is hydrolyzed to ammonia and glutamate.

2. Bicarbonate is activated by ATP phosphorylation forming
carboxyphosphate.

3. Carboxyphosphate reacts with ammonia to form carbamic

acid.
4. Carbamic acid is phosphorylated by a second ATP to form
carbamoyl phosphate.
 CPSase Structure
1. Reactions occur on different domains or subunits.
2. Active sites are far away from each other.
3. The intermediates are labile.
4. Intramolecular tunnels connect active sites.
5. Intermediates are sequestered within the molecule.
tunnels
GLN

CPS.A

CPS.B
E. coli Regulation of Pyrimidine Biosynthesis
1. There is one carbamoyl phosphate synthetase that provides
carbamoyl phosphate for both pyrimidines and arginine
biosynthesis.
2. CPSase is regulated by metabolites from both pathways.
Ornithine activates, UMP inhibits, IMP activates.
3. ATCase catalyzes the first committed step in the pyrimidine
pathway and is allosteric inhibited by CTP and activated by ATP.
Mammals
1. There are two CPSases, one for pyrimidines and
one for urea and arginine biosynthesis.
2. The pyrimidine specific CPSase is inhibited by
UTP, activated by PRPP and controlled by PKA and
MAP kinase phosphorylation.
3. ATCase is unregulated.
 Interconversion of Pyrimidines
1. Nucleoside monophosphates
are converted to diphosphates by UMP
specific nucleoside monophos- ATP nucleoside
phate kinases. ADP monophosphate kinase
2. Nucleoside diphosphates are UDP
converted to triphosphates by a XTP nucleoside
diphosphate kinase
broad spectrum enzyme, XDP
nucleoside diphosphate
kinase, that works with any pair. CTP
synthetase
3. UTP is converted to CTP by
CTP synthetase.
4. dUMP is converted to TMP by thymidylate synthase
(not as shown here).
5. CTP and other di and trinucleotides are converted to
deoxyribonucleotides by ribonucleotide reductase.
de novo Purine Biosynthesis
1. Starting point is PRPP.
2. Ring system is built upon
PRPP.
3. The pathway consists of 10
10 steps, the imidazole ring is
constructed first. The six
member ring is then built from
this intermediate.
4. The endproduct of the
pathway is inosinate (IMP).
5. IMP is converted to AMP and
GMP and ultimately ATP and
GTP.
6. The deoxypurine nucleotides
are formed by ribonucleotide
reductase.
 5- Phosphoribosyl-1- Pyrophosphate (PRPP)

1. PRPP is used for the synthesis of pyrimidines and

purines and many other metabolites.
2. PRPP is synthesized from ribose 5 phosphate and ATP.
3. The enzyme, PRPP synthetase, is allosteric regulated.
 1. The purine ring is assemble on PRPP.

2. The first step in the pathway is catalyzed by amido

phosphoribosyl transferase, an enzyme with two domains,
and is a control point.

3. Ammonia produced by the glutaminase domain of the

enzyme. Ammonia then reacts with PRPP on the synthetase
domain.
glutamine glutamate
O
- NH3 PP O
O P O CH2 NH2
-
- O O P O CH2
O O
-
O
OH
OH O -
O H
- -
OH
OH
PRPP O P O P O 5-phosphoribosyl-1-amine
O O
 Synthesis of AMP and GMP
IMP is converted to AMP in two steps.
1. aspartate is the nitrogen donor.
2. GTP is the energy donor

IMP

IMP is also converted to GMP in two steps.

1. NAD+ is the hydrogen acceptor.
2. ATP is the energy donor (the equivalent of 2 ATPs)
 Regulation of Purine Biosynthesis

Major Control Points

1. PRPP Synthesis
2. PRPP Amidotransferase
3. feedback inhibition at the AMP and GMP branch point
4. reciprocal energy source.
5. coordinate regulation of transcription by PurR repressor.

inhibited by AMP
his pyr
Phospho AMP
Ribose 5-P PRPP -ribosyl IMP
amine GMP
inhibited by inhibited by GMP
IMP, AMP, GMP
 Purinosomes

(A) When purines are available and there is no need for their
biosynthesis, the purine biosynthetic enzymes (here fused to GFP) are
dispersed.

(B) When purines are scarce, the enzymes transiently associate with
one another to form the purinosome (a metabolon) which synthesizes
purines more efficiently.

Assembly may be controlled by phosphorylation.

 active site

tyrosyl-radical
ribonucleotide reductase site
 Synthesis of Deoxyribonucleotides
O O O O O O
- -
O POPOPOCH2 base O POPOPOCH2 base
O O
O- O- O- O- O- O-

OH OH OH H

1. Replacement of the 2-hydroxyl group with a hydrogen.

2. The substrates are the nucleotide di- or triphosphates.
3. The reaction is a reduction and the ultimate reductant is
NADPH.
4. The reaction requires three different proteins.
1) Ribonucleotide reductase
2) Thioredoxin
3) Thioredoxin reductase
 Ribonucleotide Reductase
1. Consists of two R1
subunits, R1 and R2
2. R1 subunit
a. active site with
three conserved
cys and a glu.
b. two allosteric
sites
3. R2 subunit R2
a. contains a very
stable tyrosine free
radical.
b. free radical
generated by a
nearby iron center.
Mechanism of Ribonucleotide Reductase

In the first step, an electron and a

proton (H atom) are removed from
one of the cys and transferred to the
stable tyrosine radical. The process
is reversed in the last step.
 Regulation of Ribonucleotide Reductase
Allosteric effectors bind to two different
types of regulatory site.
 Thymidylate Synthase
1. Thymidylate is formed from deoxyuridylate by methylation of
C5 on the pyrimidine ring using N5, N10 methyleneTHF.
2. To make the C5 ring carbon a good nucleophile, dUMP is
conjugated at C6 to a cysteine on the enzyme.
3. The enzyme also binds methyleneTHF
in a conformation that favors the open
configuration of the 5 member ring.

4. A hydride is transferred from THF to

form the methyl group.
1. The other product of thymidylate synthase is
dihydrofolate.
2. For the reaction to proceed N5,N10 methylene THF
must be regenerated.

3. Reduced to THF by NADPH,

followed by reaction with
serine.
4. Thymidylate syntatase and
dihydrofolate reductase are
targets for chemotherapeutic
agents.
Nucleotides are also precursors of important
molecules formed from ATP.
1. NAD+
2. FAD
3. Coenzyme A