Chapter 22 1 Takusagawas Note

Chapter 22: Nucleotide Metabolism

Nucleotides are:
- monomers of RNA and DNA.
- energy sources.
- carriers of lipids and carbohydrate (such as UDP-glucose).
- form various coenzymes (such as NAD+).

Purines: A and G
Pyrimidines: C, T, and U

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 Chapter 22 2 Takusagawas Note

Synthesis of purine ribonucleotides

By feeding a variety of isotopically labeled compounds to pigeons, and determining the position
of the labeled atoms in their excreted uric acid (a purine), the biosynthetic origin of purine
ring atoms were determined.
HCO3-
O H Gly
Asp amine
H N
N C N
O N 1 6 5C 7
2 3 4 9
8C Formate
O N N C C
Formate N N
H H H
Uric acid Gln amine
- For example, C6 is come from CO2.
- The initially synthesized purine derivative is inosine phosphate (IMP), its base is
hypoxanthine.

O
N Hypoxanthine base
HN

O N N
-
O P O O
-
O

OH OH
Inosine monophosphate (IMP)

IMP synthesis
1. Formation of phosphoribosylpyrophosphate (PRPP) from ribose-5-phosphate (R5P) by ATP.
2. Replacement of PPi with NH2 of glutamine [N9].
3. Attachment of glycine to the NH2 group to produce glycinamide ribotide (GAR) [C4, C5, N7].
4. Attachment of formyl group from N10-formyl-THF [C8]. GAR transformylase that catalyzes
this reaction is a target of anti-cancer drug since to stop synthesis of purine is to stop DNA
synthesis.
5. Attachment of NH2 from glutamine to C4 [N3].
6. Formation of purine imidazole ring by ring close.
7. Attachment of CO2 (HCO3-) to C5 [C6].
8. Attachment of Asp to the C=O [N1].
9. Release fumarate from Asp. Only NH2 group remains on C=O.
10. Attachment of formyl group from N10-formyl-THF [C2].
11. Cyclization to form IMP.

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Chapter 22 3 Takusagawas Note

2-
O3P

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 Chapter 22 4 Takusagawas Note

Note: technical term of de novo synthesis --- biosynthesis from entirely small molecules,
such as IMP that synthesized from PRPP, amino acids (Gln, Gly, Asp), CO2 and N10-formyl-
THFs.

In the IMP synthesis,

- Gln and Asp donate -NH2 at specific reaction steps.
- N10-formyl-THF involved in two steps.
- IMP is the product that has hypoxanthine base.

Synthesis of adenine and guanine ribonucleotides from IMP

- AMP --- Asp donates -NH2 to C6 position of IMP.

- GMP --- Gln donates -NH2 to C2 position of IMP.

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 Chapter 22 5 Takusagawas Note

Di and tri-phosphate nucleotide synthesis

Di- and tri-phosphate nucleotides are synthesized from mono-phosphate nucleotides by catalyzed
by nucleoside monophosphate kinase and nucleoside diphosphate kinase, respectively.

nucleoside monophosphate kinase

NMP + ATP NDP + ADP
e.g., GMP + ATP GDP + ADP and AMP + ATP 2ADP

nucleoside diphosphate kinase

NDP + ATP NTP + ADP
e.g., GDP + ATP GTP + ADP

Salvage of purines
- Many of nucleic acids are degraded and this process releases adenine, guanine, and
hypoxanthine.
- These bases are reused to produce nucleotides by following salvage pathways.

adenine phosphoribosyltransferase (APRT)

Adenine + PRPP AMP + PPi

hypoxanthine - guanine phosphoribosyltransferase (HGPRT)

Hypoxanthine + PRPP IMP + PPi

hypoxanthine - guanine phosphoribosyltransferase (HGPRT)

Guanine + PRPP GMP + PPi

- Lack of HGPRT results in Lesch-Nyhan Syndrome (poor muscle control, and moderate
mental retardation).
- Since lack of salvage pathway, purines must synthesized by de novo processes (thus, de
novo processes increase).
- Purine concentration is increased because no salvage process. Therefore, too much
resulting purines leads to uric acid production.
- The gene is carried by the mother and passed on to her son.

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 Chapter 22 6 Takusagawas Note

Regulation of purine nucleotide biosynthesis (Mainly feedback inhibitions)

- PRPP activates formation of 5-phosphoribosylamine (PRamine).
- ADP & GDP inhibit synthesis of PRPP from ribose-5-phosphate (R5P)
- All purine nucleotides (AMP, ADP, ATP, XMP, GMP, GDP, GTP) inhibit formation of
PRamine
- AMP & GMP inhibit their synthesis from IMP.
- ATP stimulates GMP synthesis as the substrate, similarly GTP stimulates AMP synthesis,
i.e., high [ATP] is reduced by using ATP to synthesize GTP, whereas, high [GTP] is reduced
by using GTP to synthesize ATP.

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 Chapter 22 7 Takusagawas Note

Synthesis of pyrimidine ribonucleotides

- Unlike purine synthesis, the base is synthesized, and then PRPP is attached.
- The first base made is orotic acid.
- Pyrimidine ring is synthesized from Asp, HCO3- and Gln (de novo synthesis).
O
H C
N Gln N 3 4 5C Asp
-
O N COOH HCO3 C 2 1 6C
N
H
Orotic acid

UMP synthesis is given below: Cytosolic

enzyme

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 Chapter 22 8 Takusagawas Note

1. Formation carbamyl phosphate from HCO3- and glutamine with enzyme carbamyl phosphate
synthetase II [C2 from HCO3-, N3 from Gln]. (Note, this enzyme is a cytosolic enzyme
whereas the enzyme in urea cycle is a mitochondrion, and use ammonia as its nitrogen
source).
2. Addition of aspartate (Asp) [C4, C5, C6, N1].
3. Cyclization by forming an amide bond.
4. Oxidation by using quinone to form orotate.
5. Addition of PRPP.
6. Decarboxylation to form UMP.

Di- and tri-phosphate nucleotides from UMP by kinases

UMP + ATP UDP + ADP
UDP + ATP UTP + ADP

CTP is formed from UTP

- NH2 is donated from glutamine.

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 Chapter 22 9 Takusagawas Note

Regulation of pyrimidine nucleotide biosynthesis

Animals --- Carbamyl phosphate synthetase II (CPS II) is the major control enzyme.
- Note: CPS II is a cytosol enzyme, whereas CPS in urea cycle is a mitochondrial enzyme.
- CPS II is activated by ATP and PRPP, and inhibited by products UTP & CTP (feedback
inhibition).
- UMP inhibits own synthesis from OMP.

Bacteria --- Aspartate transcarbamoylase (ATCase) is the major control enzyme.

- ATCase is activated by Gln and ATP.
- ATCase is inhibited by the produce CTP (feedback inhibition).

Carbamyl phosphate synthetase II

(CPS II)

Aspartate transcarbamoylase
(ATCase)

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 Chapter 22 10 Takusagawas Note

Formation of deoxyribonucleotides
- Deoxyribonucleotides are synthesized from their corresponding ribonucleoties by the
reduction of C2 position.

RNA

DNA

Ribonucleotide reductases are key enzymes because they involved in DNA synthesis.
- Substrate is NDP (such as ADP, GDP, CDP, etc.).
- There are several types of reductase.
- Class I --- All eukaryotes and many prokaryotes.
- Functional group is a tyrosyl radical stabilized by an oxo-bridged binuclear Fe(III)
complex, Fe(III) O2-Fe(III).
- Class II --- Only in prokaryotes
- Use coenzyme B12.
- Class III --- In anaerobic prokaryotes
- contains Fe-S cluster and use SAM (S-adenosylmethionine)

- Class I reductase composes of 2 pairs of dimer, R1(2)R2(2).

ADP

- -subunit contains the tyrosyl radical and Fe-O-Fe complex.

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 Chapter 22 11 Takusagawas Note

Note: radical = unpaired electron.

H

OH O

Tyrosyl radical

Note: the bridge oxygen is O2- (that has two additional electrons), but not O2.

- -subunit has two different allosteric sites.

1. Control the enzyme activity (activity site)
2. Control the substrate specificity (specificity site)
These activity and specificity sites regulate the reductase activity, which described later.
- -subunit contains important -SH groups.

- Catalytic site is located interface between and -subunits.

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 Chapter 22 12 Takusagawas Note

Reaction mechanism

Enzyme is oxidized

1. The tyrosyl radical abstracts H-atom from C3.

2 & 3. Acid-catalyzed cleavage of C2-OH yields radical-cation intermediate.
4. E-SH reduces the cation (by hydride transfer), and the oxidized sulfhydryl pair form disulfide
group -S-S-.
5. The radical intermediate receives H-atom from the tyrosine to its C3 position, resulting the
product, and tyrosyl radical and disulfide group in the enzyme.
6. The disulfide group is reduced to -SHs by use of thioredoxin (ubiquitous monomeric 108-
residue protein). Electrons are provided from NADPH molecules.
Regeneration of -SH groups in the reductase

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 Chapter 22 13 Takusagawas Note

- Ribonucleotide reductase, thioredoxin, and thioredoxin reductase have sulfhydryl pair that
forms disulfide bond by oxidation.

SH S
Thioredoxin + Ribonucleotide reductase
(reduced) SH S (oxidized)

S HS
Thioredoxin + Ribonucleotide reductase
(oxidized) S HS (reduced)

1. Oxidized -S-S- of ribonucleotide reductase is reduced by thioredoxin.

2. Oxidized -S-S- of thioredoxin is reduced by thioredoxin reductase.
3. Oxidized -S-S- of thioredoxin reductase is reduced by FADH2.
4. FAD (oxidized FADH2) is reduced by NADPH.
5. NADP+ (oxidized NADPH) is reduced by the pentose phosphate pathway.

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 Chapter 22 14 Takusagawas Note

Regulation of ribonucleotide reductase

- R1 (2) subunit has activity site and specificity site.

- Activity site:
- ATP binding activates the enzyme.
- dATP binding inhibits the enzyme.

- Specificity site:
- ATP binding stimulates the CDP & UDP reductions.
- dTTP binding stimulates the GDP reduction, but inhibits the CDP & UDP reductions.
- dGTP binding stimulates ADP reduction, but inhibits the CDP, UDP & GDP reductions.

CDP dCDP dCTP = Inhibit

ATP
UDP dUDP dTTP = Activate

GDP dGDP dGTP

ADP dADP dATP

- All dinucleotides (NDP) are present in cells. Thus, NDPs are reduced to dNDP if there is
not proper regulation system.
- The regulation of the reductases can be interpreted as follows:
1. Relatively large amount of ATP is present in cells. The ATP stimulates the reduction of
pyrimidine nucleotides (CDP & UDP).
2. As a feedback mechanism, dTTP inhibits the reduction of pyrimidine nucleotides, and
stimulates the reduction of GDP.
3. As a feedback mechanism, dGTP inhibits the reduction of CDP, UDP, and GDP, and
stimulates the reduction of ADP.
4. As a feedback mechanism, dATP inhibits the reduction of all NDPs unless [ATP] is
sufficiently high.

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 Chapter 22 15 Takusagawas Note

Thymidylate synthesis
- dTMP is synthesized from dUMP by thymidylate synthase with N5, N10-methylene-THF as
the methyl donor.

Oxidized form

- In the reaction, not only CH2 group is transferred from the N5, N10-methylene-THF, but also
an electron is transferred. Thus, N5, N10-methylene-THF is oxidized to dihydrofolate.
- Therefore, the thymidylate synthesis involves both methyl-transfer and reduction-oxidation
reaction.
- Details of the reaction is shown in Fig. 26-18.
1. Cys 146 attacks C6 of dUMP to form a covalent adduct.
2. Formation of enzyme -dUMP-THF ternary covalent complex.
3. The H-atom attached to C5 of dUMP is removed by base-catalysis of the enzyme and the
methylene group of N5, N10-methylene-THF is transferred to C5 of dUMP.
- Note: A potent antitumor agent, 5-fluorodeoxyuridylate (FdUMP), inhibits this step since the
H5 of dUMP is a relatively good leaving group, but the F-atom is not. Thus, if FdUMP
enters into the active site as a substrate, the dTMP synthesis is completely stopped.
Therefore, FdUMP is called a dead-end compound.

O
H F Bad leaving group
N

O O N
-
O P O O
-
O

OH
5-Fluorodeoxyuridylate (FdUMP)
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 Chapter 22 16 Takusagawas Note

4. Transfer H-atom at C6 of THF to the methylene group at C5 of dUDP, and cleavage of

enzyme-dUMP bond to produce dTMP.

Ternary complex
E-dUMP-THF

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 Chapter 22 17 Takusagawas Note

Note: Both dUTP and dTTP are taken into the DNA synthesis without any discrimination.
- Thus, it is important to keep [dUTP] to be very low.

dUTP diphosphohydrolase
dUTP + H2O dUMP + PPi

- Therefore, dUTP diphosphohydrolase is a very important enzyme.

Regeneration of THF

- As mentioned, thymidylate synthase reaction is biologically unique since THF is oxidized to

DHF (net oxidized state).

- N5, N10-methylene-THF is regenerated by two reactions.

1. DHF THF --- catalyzed by dihydrofolate reductase (DHFR) with NADPH.
Dihydrofolate reductase is strongly inhibited by methotrexate, aminopterin and trimethoprin,
analogues of THF. Methotrexate is a potent anti-cancer drug.
2. THF N5, N10-methylene-THF --- catalyzed by serine hydroxymethyl transferase with
serine as substrate and glycine as product.

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 Chapter 22 18 Takusagawas Note

Nucleotide degradation
Purines
- Purines are metabolized to uric acid.
- Two kinds of enzymes are involved in this metabolism.
nucleotidase
1. Nucleotide + H 2 O nucleoside + Pi
nucleosidase
2. Nucleoside + H 2 O base + ribose
and / or
nucleoside phosphorylase
Nucleoside + Pi base + ribose 1 P

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 Chapter 22 19 Takusagawas Note

- Gout is a disease characterized by elevated levels of uric acid in body fluids.

- deposition of insoluble sodium urate in cartilage of joints, and kidney and ureter as
stones.
- Allopurinol is a structural analogue of hypoxanthine, and is a gout treatment drug that
inhibits xanthine oxidase. Thus, [uric acid] in body fluids is decreased.
O O
N
HN HN
N
N N N
N H
H
Allopurinol Hypoxanthine

- Allopurinol has been used for minor Lesch-Nyhan Syndrome treatment by reducing
purines in body fluids

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 Chapter 22 20 Takusagawas Note

Pyrimidines
- Pyrimidines are metabolized to malonyl-CoA from CMP and UMP and to methylmalonyl-
CoA from dTMP, i.e., unlike purine metabolism, the pyrimidine rings are opened.
- Malonyl-CoA is precursor of fatty acid synthesis, and methylmalonyl-CoA is a product of
odd-chain fatty acid degradation and is converted to succinyl-CoA and enters into the citric
acid cycle.

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