biosint nucleotidos

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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.

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

Purine Nucleotide Biosynthesis

Purine biosynthesis. Steps 1 - 2 Precursors PRPP + Gln The purine ring is built on top of the ribose-5-phosphate.


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Purine biosynthesis. Steps 3 - 4

Folate is a source of carbon






Purine biosynthesis. Steps 9 - 11 Up to the formation of inosinate, IMP, 5 ATPs are consumed.


Formation of AMP and GMP from IMP. One additional GTP/ATP consumed per product.


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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Regulation of purine biosynthesis.!!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. !


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.


Pyrimidine biosynthesis. Precursors: HCO3

-, 2ATP, glutamine, PRPP The pyrimidine ring is built first, and then attached to PRPP. Steps 1 - 5

HCO3- + 2ATP + glutamine glu + P i

Pyrimidine Nucleotide Biosynthesis

carbamoyl phosphate synthetase

Pyrimidine biosynthesis. 6 steps to form UMP. Additional steps required to form CTP.


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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bacteria animals

carbamoyl phosphate

CPS (general)

ATCase urea cycle carbamoyl phosphate

WHERE TO REGULATE PYRIMIDINE BIOSYNTHESIS???

UMP

CPS II (dedicated)

ATCase

UMP


In bacteria, the pathway is regulated at step 2, by allosteric regulation of aspartate transcarbamoylase

Sigmoidal saturation curves are typical of allosterically regulated enzymes. K0.5 is the [S] at 1/2 Vmax.


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

Kinases

Deoxyribonucleotides (for DNA) are derived from the corresponding ribonucleotides.

Reaction catalyzed by ribonucleotide reductase (RR).

Formation of deoxyribonucleotides

Source of electrons: glutaredoxin (GSH) and thioredoxin (FADH2). GSH and FADH2 are reduced by NADPH.

Note: nucleotide diphosphates are the substrates for the reaction.


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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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

Thymidylate Synthase. The methyl group of dTMP originates from tetrahydrofolate. Tetrahydrofolate is an enzyme cofactor involved in transfer of 1-carbon units.

Thymidylate synthase

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

Purine Degradation. Genetic deficiencies in adenosine deaminase result in severe immunodeficiency. Patients must live in a sterile environment.


Pyrimidine Degradation. The pathways generally lead to formation of NH4+.


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 (automutilación y retardo mental).

Salvage pathways

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Gout (gota) 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

Allopurinol inhibits xanthine oxidase.


“Pyrimidine biosynthesis is invariably up-regulated in tumors and neoplastic cells.”

Evans and Guy J Biol Chem. (2004) PMID: 15096496

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

Cancer Chemotherapy


Cancer Chemotherapy Chemotherapeutic agents that inhibit enzymes of nucleotide synthesis. Example: Glutamine analogs.


Inhibition of dTMP production by FdUMP, methotrexate, and aminopterin.


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