bmsc 230 module 13 nucleotide metabolism

What is a nucleotide?

-Sugar
-Base
-Phosphate(s)

What is a nucleoside?

Base attached to sugar.

NO phosphates.

What are the purines?

Have double rings.

Adenine and guanine.

What are the pyrimidines?

Single rings.

Cytosine, thymine, uracil.

What differs between uracil and thymine?

Presence of a methyl group in thymine.

Name the five nucleosides. The five nucleotides.

A:
-Adenosine
-Adenylate

G:
-Guanosine
-Guanylate

U:
-Uridine
-Uridylate

C:
-Cytidine
-Cytidylate

T:
-Thymidine
-Thymidylate

Describe the two pathways to synthesize nucleotides.

1. De Novo Pathway:
   -Made from PRPP, amino acids, ATP, CO2
   -Must synthesize the base.

2. Salvage Pathway:
   -Made from PRPP and base.

Describe the basic strategy behind the de novo synthesis of pyrimidines.

1. Synthesize carbamoyl phosphate from bicarbonate (CO2) and ammonia (Gln).
2. Build orotate (pyrimidine ring) out of carbamoyl phosphate, and aspartate.
3. Link orotate to PRPP to form OMP.
4. Convert OMP to UMP.

What is PRPP?

5-phosphoribosyl-1-pyrophosphate

Describe the synthesis of carbamoyl phosphate used for nucleotides.

Carbamic acid is made from bicarbonate and ammonia (from Gln).

Catalysed by CPSII (carbamoyl phosphate synthetase II).
-Uses ATP

Carbamic acid is phosphorylated using ATP to carbamoyl phosphate.

How do CPSI and CPSII differ?

Process:
I: Urea synthesis
II: Pyrimidine base synthesis

Location:
I: Mitochondrial matrix of liver
II: Cytosol of many tissues

Nitrogen donor:
I: NH4+
II: Gln (NH3)

How is CPSII regulated?

(-) by CTP and UTP

(+) by ATP

How is CPSI regulated?

(+) by N-acetylglutamate and Arginine

Describe the three reaction formation of orotate.

[1] Carbamoyl group is transferred from carbamoyl phosphate to aspartate to produce carbamoylaspartate.

This (1) is catalysed by aspartate transcarbamoylase.
-Reversible

[2] Carbamoylaspartate cyclizes to form dihydroorotate.

This (2) is catalysed by dihydroorotase.

[3] Dihydroorotate is then oxidized by NAD to orotate.
-Produces NADH.

How are CPSII, aspartate transcarbamoylase and dihydroorotase related?

All involed in synthesis of orotate.

All encoded by one gene (CAD) and synthesized on one peptide.

IN MAMMALS.

Describe the two reaction formation of uridylate from orotate.

Orotate reacts with PRPP to form orotidylate.
-Catalysed by orotate phosphoribosyltransferase.

Orotidylate is decarboxylated to form uridylate (UMP).
-Catalysed by orotidylate decarboxylase
-Produced CO2

What is uridine monophosphate synthetase?

Bifunctional enzyme containing phosphoribosyltransferase and orotidylate decarboxylase.

These are both needed to convert orotate to UMP.

Both encoded by one gene (hence, on the same peptide).

What happens to UMP as it is synthesized?

It is converted first to UDP and then to UTP.

UTP can be converted to other pyrmidines.

Describe how CTP is formed.

Amination of UTP.

Carbonyl oxygen of UTP is replaced with an amino group by cytidine triphosphate synthetase.
-Amino group is derived from Gln.

How is aspartate transcarbamolase regulated?

Inhibited by CTP.
-CTP is the final product of the pathway of pyrimidine synthesis.

Stimulated by ATP.
-This is a purine, activation by purines serves to balance the two nucleotide pools.

Describe how dTMP is synthesized via the salvage pathways.

Thymine is released from a nucleotide and absorbed in to the body.

Thymine is converted to the nucleoside deoxythymidine by reaction with deoxyribose-1-phosphate.
-Catalysed by thymidine phosporylase.

Deoxythymidine is then converted to the nucleotide dTMP.
-Catalysed by thymidine kinase.
-Uses ATP.

Describe the general strategy behind de novo synthesis of purines.

1. Assembled onto the ribose by first attaching ammonia.
2. Build imidazole ring with glycine, formyl group and ammonia (from Gln).
3. Second ring is built from aspartate, bicarbonate, formyl group and ammonia (from Gln). This forms IMP.
4. IMP is converted to AMP and GMP.

Describe the first step of de novo purine synthesis.

Ammonia (from Gln amide nitrogen) displaces the pyrophosphate of PRPP to form 5-phosphoribosyl-1-amine.
-Catalysed by glutamine phosphoribosyl amidotransferase.

Which amino group is donated by Gln?

Amide nitrogen.

Describe the four steps to assemble the first ring of a purine. The five steps of the second ring?

First Ring:
[1]: Glycine is coupled to amino group.
[2]: Formyl group is donated by N10-formylH4Folate to the amino group of glycine.
[3]: Carbonyl of inner amide is phosphorylated and the ammonia (from Gln) displaces it to form an amidine.
[4]: Intramolecular reaction forms five-membered ring

Second ring:
[1]: Carboxyl group from bicarbonate is added to the exocycylic amino group. Then, it is transferred to carbon 4.
[2]: Imidazole COO is phosphorlated and phosphate is displaced by aspartate.
-Produces ATP.
[3]: Fumarate leaves (leaving only an amino group from Asp).
[4]: Formyl group from N10-formylH4Folate is added to the amino group from Asp.
[5]: Intramolecular cyclisation forms inosine monophosphate (IMP).

What is the base of inosine?

Hypoxanthine.

What is required for AMP synthesis? GMP synthesis?

AMP requires GTP.

GMP requires ATP.

Describe how AMP is made from IMP.

Carbonyl group of IMP is phosphorylated and phosphate is displaced by aspartate.
-Forms adenylosuccinate.
-Uses GTP.

Fumarate leaves and this leaves only an amino group from Asp on Inosine.

Aminated (at this position) inosine is adenosine.

Describe how GMP is made from IMP.

Water is added to IMP to add another carbonyl group.
-Uses NAD (oxidation)
-Forms xanthylate

ATP is used to phosphorylate new carbonyl and then it is replaced with an amino group (from Gln).

This aminated (at this position) inosine is guanosine.

How is glutamine phosphoribosyl amidotransferase regulated?

Inhibited by several ribonucleotides.

Notably:
-AMP
-GMP

How is AMP synthesis regulated?

AMP inhibts formation of adenylosuccinate.

GTP stimulates formation of adenylosuccinate.

How is GMP synthesis regulated?

GMP inhibits the formation of xanthylate.

ATP stimulates formation of xanthylate.

Describe the two salvage pathways of purine synthesis.

1. Adenine displaces PP of PRPP to form AMP.
   -Catalysed by adenine phosphoribosyltransferase.

2. Guanine displaces PP from PRPP to form GMP.
   -Catalysed by hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
   -Can also use hypoxanthine to form inosinate (IMP).

How is hypoxanthine produced?

Deamination of adenine.

How are monophosphates converted into diphosphates?

Nucleoside monophosphate kinases use ATP to add a phosphate to an NMP to form a NDP.

This is reversible.
-Forms ADP.

How are nucleoside monophosphate kinases specific?

Specific to the base of the NMP being converted to a NDP.

How are diphosphates converted to triphosphates?

Nucleoside dipshosphate kinase (non specific for sugar and base) catalyses:

XDP + YTP to XTP + YDP

YTP is usually ATP.
-This reaction is also reversible.

Why is ATP typically used by Nucleoside dipshosphate kinase?

It is present in the highest [] compared to other NTPs.

Describe how ribonucleotides are converted to deoxribonucleotides.

Made from ribonucleotide diphosphates.

2' OH is replaced with hydrogen.
-Catalysed by ribonucleotide reducates (for all ribonucleotides).

Describe how the ribonucleotide reductase works.

In the final step of its reaction two SH groups in the protein are oxidized into S-S.

This reduces the 2' OH group of a ribonucleoside diphosphate to H.

This forms a dioxyribonucleotide diphosphate.

What are the three components involved in reduction of ribonucleotide diphosphates?

1. Thioredoxin reductase
   -Accepts NADPH with FAD
   -Reduces thioredoxin SH groups.

2. Thioredoxin
   -Reduces ribonucleotide reductase SH groups.

3. Ribonucleotide reductase
   -Site of ribonucleotide reduction.

Each contains two SH groups which shuttle electrons from NADPH to the reductase site to reset (re-reduce) it after each reduction.

Where do the electrons to reduce ribonucleotide reductase come from?

NADPH.

Accepted by thioredoxin reductase and passed through thioredoxin to ribonucleotide reductase.

How is deoxythymidylate formed?

dUMP is made from UMP.

dUMP is methylated using N5N10-methylenetetrahydrofolate to form dTMP.
-Catalysed by thymidylate synthase (a methyl transferase).

How is dTMP synthesis targeted to treat cancer?

Especially important for DNA syntheiss (such as in rapdily dividing cancer cells).

Drugs (such as fluorouracil) can form covalent complexes with thymidylate synthase and stop its activity permanently.

Drugs can also target dihydrofolate reductase.
-This is needed to produced tetrahydrofolate which is a cofactor of thymidylate synthase.

What are two dihydrofolate reductase inhibitors?

Aminopterin.

Methotrexate (amethopterin).

Both have MUCH higher affinities for dihydrofolate reductase than dihydrofolate.

How does fluorouracil work?

Uracil with a fluorine on it.

Thymidylate synthase recognises fluorouracil instead of uracil of UMP.

Forms covalent bond with fluorouracil that cannot be broken (fluorine is in the site where methylation should usually occur). This stops all enzymatic activity.

Describe the degredation of purines.

[G] Guanine is degraded to xanthine.

[A] AMP is dephosphorylated to form adenosine.
-Catalysed by nucleotidase

Adenosine is deaminated to form inosine.
-Catalysed by adenosine deaminase.
-Uses H2O

Inosine has ribose removed to form hypoxanthine.
-Catalysed by nucleoside phosphorylase.

Hypoxanthine is oxidized to form xanthine.
-Catalysed by xanthine oxidase.
-Uses O2 and H2O and produces H2O2.

Xanthine is further oxidized by xanthine oxidase to uric acid.
-Catalysed by xanthine oxidase.
-Uses O2 and H2O and produces H2O2.

Uric acid is converted to sodium urate which is excreted.

What do RNAase and DNAase do?

Cleave bonds between individual nucleotides in RNA and DNA.

What do nucleotidases do?

Cleave phosphates off of nucleotides to form nucleosides.

What do nucleoside phosphorylases do?

Cleave bond between sugar and base of a nucleoside.

Describe severe combined immunodeficiency.

One form is due to the loss of adenosine deaminase (needed for adeonisine degredation).

Increase adenosine interrupts signalling pathways.
Also, high dATP inhibits ribonucleotide reductase (stopping DNA synthesis).

This lead to few or no T cells and non-functional B cells (no immune function).

How is ribonucleotide reductase regulated?

Inhibited by dATP.

Describe gout.

AKA gouty arthritis.

High levels of blood uric acid are caused by overproduction and underexcretion of uric acid.

Sodium urate crystalises in the joints, causing pain and inflammation.

Treated by:
-Drugs to reduce production of uric acid (inhibit xanthine oxidase).
-Excretion stimulants.

What is allopurinol?

Drug that is converted to oxypurinol by xanthine oxidase.

Oxypurinol binds the enzyme tightly and stops its activity.

Describe lesch nyhan syndrome.

Caused by defective hypoxanthine-guanine phosphoribosyltransferase.
-Part of purine salvage pathway for guanine and inosine.

X linked, rare in females.
-Very rare in general.

Causes:
-High urate levels (gout, kidney problems)
-Poor muscle control, intellectual disability
-Self destructive behaviours

Why does the absence of HGPRT cause abnormal behaviour?

Not clear. Two theories:

1. Brain has limited de novo purine synthesis. Therefore, purines are not synthesized in high enough amounts when salvage pathways are interrupted.
2. May cause imbalanced neurotransmitters or interfere with G protein function.

Why is the addition of orotate to PRPP irreversible?

PP is rapidly hydrolysed.