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MBIO 2100 




Lehninger Principles of Biochemistry, 4th ed.


CHAPTER 22 (pp. 862 - 876)

(Chapters 22, pp. 848 - 865, 3rd ed.)

Biosynthesis and Degradation of

Nucleotides





Text and figures from Lehninger 3rd ed. or 4th ed.

The pools (reservas) of nucleotides

in cells are < 1% of the quantities
required for DNA synthesis.

How does the cell get the

nucleotides it needs?



Synthesis pathways

Salvage (reciclaje) pathways

Synthetic pathways

Identical in nearly all organisms

Free bases are NOT pathway intermediates

Precursors -

Source of N: Gln, NH3

PRPP (from ribose-5-phosphate)

Gly, Asp, Formate (purines)

Asp (pyrimidines)

HCO3-

Salvage pathways

recycle free bases and nucleosides from nucleic
acid degradation.

Purine Nucleotide Biosynthesis

Purine Nucleotide Biosynthesis

Purine biosynthesis.



Steps 1 - 2



Precursors

PRPP + Gln



The purine ring is

built on top of the

ribose-5-phosphate.

Origin ring atoms determined by Buchanon (1950s)
using 14C- and 15N-labeled precursors in birds.

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Purine Nucleotide Biosynthesis

Purine Nucleotide Biosynthesis

Purine biosynthesis.

Purine biosynthesis.

Steps 3 - 4

Steps 5 - 6

Folate is a
source of
carbon

Purine Nucleotide Biosynthesis

Purine Nucleotide Biosynthesis

Purine biosynthesis.

Steps 9 - 11



Up to the formation

of inosinate, IMP,

5 ATPs are consumed.

Purine biosynthesis.

Steps 7 - 8

Purine Nucleotide Biosynthesis

Formation of AMP and GMP from IMP.

One additional GTP/ATP consumed per product.

Purine Nucleotide Biosynthesis

Regulation of
purine biosynthesis.!
!
1(a) The end product
ADP inhibit the
synthesis of the
precursor PRPP.!
!
1(b) The first unique
step in the pathway is
feedback-inhibited by
the end products IMP,
GMP, AMP.!

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Purine Nucleotide Biosynthesis

Purine Nucleotide Biosynthesis

Regulation of
purine
biosynthesis.!

Regulation of
purine
biosynthesis.!
!
3. GTP is required for
the synthesis of AMP
from IMP. ATP is
required for the
synthesis of GMP
from IMP.!
!
This level of control
balances the synthesis
of AMP and GMP.

!
2 (a) An excess of
GMP inhibits the
formation of GMP
from IMP.!
!
2 (b ) An excess of
AMP inhibits the
formation of AMP
from IMP. !

Pyrimidine Nucleotide Biosynthesis

Pyrimidine Nucleotide Biosynthesis

glu + P i

carbamoyl phosphate synthetase

HCO3- + 2ATP + glutamine

Pyrimidine
biosynthesis.



6 steps to form UMP.



Additional steps
required to form CTP.

Pyrimidine biosynthesis.

Precursors:

HCO3-, 2ATP, glutamine,
PRPP



The pyrimidine ring is

built first, and then
attached to PRPP.



Steps 1 - 5

Pyrimidine Nucleotide Biosynthesis

Pyrimidine Nucleotide Biosynthesis

Pyrimidine biosynthesis in animals and bacteria

animals:

2 separate enzymes produce carbamoyl phosphate. Carbamoyl
phosphate synthetase II (CPSII) is the enzyme in pyrimidine
biosynthesis. What other pathway uses a CPS?

Step 1 is regulated (CPSII)

Steps 1 - 3 are catalyzed by a large, multifunctional enzyme

called CAD.

bacteria:

Only one enzyme produces carbamoyl phosphate for both
pathways.

Step 2 is regulated (aspartate transcarbamoylase)

Steps 1 - 3 are catalyzed by monofunctional enzymes.

Urea cycle associated carbamoyl synthetase I (CPS I) in

mammals.



Bacteria have only one CPS that is used for both urea
cycle and pyrimidine biosynthesis.

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Pyrimidine Nucleotide Biosynthesis

bacteria

Pyrimidine Nucleotide Biosynthesis

animals

CPS (general)

carbamoyl phosphate

urea cycle

In bacteria, the pathway is regulated at step 2, by

allosteric regulation of aspartate transcarbamoylase

CPS II (dedicated)

Sigmoidal saturation curves are typical of allosterically regulated

enzymes. K0.5 is the [S] at 1/2 Vmax.

carbamoyl phosphate

ATCase

UMP

ATCase

UMP

WHERE TO REGULATE PYRIMIDINE BIOSYNTHESIS???

Kinases

Formation of deoxyribonucleotides

Nucleoside monophosphates are converted

to nucleoside triphosphates.





















glycolysis or oxid. phosphoryl.

1 adenylate kinase








ATP + AMP <> 2ADP > > > > > ATP



2. nucleoside monophosphate kinases


ATP + NMP <> ADP + NDP



3. nucleoside diphosphate kinase

NTP + NDP <> NDP + NTP

Formation of deoxyribonucleotides

Deoxyribonucleotides (for DNA) are derived from the

corresponding ribonucleotides.

Reaction catalyzed by ribonucleotide reductase (RR).

Formation of deoxyribonucleotides

Note:

nucleotide
diphosphates are the
substrates for the
reaction.

Source of electrons:



glutaredoxin (GSH) and
thioredoxin (FADH2).



GSH and FADH2 are
reduced by NADPH.

Ribonucleotide reductase activity is regulated.

Regulation of enzyme s activity (primary regulation site)

Regulation of enzyme s substrate specificity (specificity site)

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Thymidylate synthase

Thymidylate synthase

Formation of Thymidylate.

DNA contains thymine rather than uracil.

The precursor of dTMP is

dUTP.

In the cell [dUTP] is low to

prevent incorporation into DNA.

Thymidylate

Synthase.



The methyl group of

dTMP originates

from tetrahydrofolate.



Tetrahydrofolate is an
enzyme cofactor involved
in transfer of 1-carbon
units.

Degradation pathways

Degradation pathways

Purine Degradation.



Genetic deficiencies in

adenosine deaminase
result in severe
immunodeficiency.

Patients must live in a
sterile environment.

Purine Degradation.



Purines are degraded to

xanthine.



In humans, xanthine oxidase

converts xanthine into

uric acid, which is excreted.



(primates, reptiles, aves, insectos)

Degradation pathways

Pyrimidine Degradation.



The pathways generally

lead to formation of
NH4+.

Salvage pathways

Salvage (Recycling) Pathways

Free purines and pyrimidines from catabolism of
nucleotides are salvaged (recycled).





Adenosine phophoribosyl transferase

Adenine + PRPP > AMP + PPi



Hypoxanthine-guanine phophoribosyl transferase
(HGPRTase)

Hypoxanthine/Guanine + PRPP > IMP/GMP + PPi

Genetic deficiency causes Lesch-Nyhan syndrome

(automutilacin y retardo mental).

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Drug targets in nucleotide metabolism

Drug targets in nucleotide metabolism

Gout (gota)

Allopurinol
inhibits
xanthine
oxidase.

Elevated blood [uric

acid], possibly due to
deficiencies in purine
metabolism.



Excess uric acid is
deposited in the joints,
kidney.



Treatment:

xanthine oxidase

Inhibitor, allopurinol

Drug targets in nucleotide metabolism

Pyrimidine biosynthesis is invariably upregulated in tumors and neoplastic cells.

Cancer cells grow more rapidly than normal cells,
and have a greater requirement for nucleotide
precursors of DNA and RNA.

Drug targets in nucleotide metabolism

Cancer Chemotherapy

Chemotherapeutic agents that

inhibit enzymes of nucleotide
synthesis.



Example: Glutamine analogs.

Cancer Chemotherapy



Evans and Guy

J Biol Chem. (2004) PMID: 15096496

Drug targets in nucleotide metabolism

Inhibition of dTMP production by

FdUMP, methotrexate, and aminopterin.