Nucleotide Metabolism

Nucleotide

1. nitrogenous base

2. pentose sugar

3. one or more phosphates

Nucleoside

1. Nitrogenous base

2. Pentose sugar

Nucleobase

Nitrogenous base

(Purine or pyrimidine)

Hypoxanthine

constituent of nucleic acids (tRNA) in form of its nucleotide, inosinate

Xanthine

produced during purine synthesis and degradation

What are the attachment points between the nitrogenous base and pentose sugar in a nucleoside?

N-9 of purine

N-1 of pyrimidine

C-1' of sugar

T/F: phosphates are attached to the base in nucleotides

False. NEVER. Phosphate attaches to the pentose sugar, to either C-2', C-3', or C-5'

Nucleobases

Adenine, guanine, uracil, cytosine, thymine

RNA ribonucleosides

adenosine, guanosine, uridine, cytidine

Deoxyribonucleosides (DNA)

deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine

Ribonucleotides

AMP, GMP, UMP, CMP

Deoxyribonucleotides

dAMP, dGMP, TMP, dCMP

purines

adenine, guanine

pryimidines

cytosine, thymine, uracil

Nucleoside nomenclature

replace -ine of purine with -osine (ex: guanosine)

replace end of pyrimidine with -idine (ex: deoxythymidine)

use deoxy- but omit ribo-

How to name 5'-monophosphates (acid salts)

add -ylate or -ate

adenylate, guanylate, cytidylate, thymidylate, uridylate

inosinate, orotate

Describe the structure of 2'-deoxycytidine 5'-triphosphate

cytosine base added to C-1' of sugar, H (not OH) bound to C-2', and 3 phosphate added to C-5'

2 methods of biosynthesis of nucleotides

1. de novo

2. salvage

What is the de novo pathway?

from new; synthesis from metabolic precursors and requires ATP

What is the salvage pathway?

Recycle free bases and nucleosides released from nucleic acid breakdown and does not require much energy

T/F: nucleotide pools are kept low in de novo biosynthesis

True. Most cells continually synthesize them because of that.

T/F: De novo synthesis may limit rates of transcription and replication

True

Where does ribose-5-phosphate come from?

Pentose phosphate pathway, breaking down glucose-6-phosphate

PRPP synthetase

converts Ribose 5-phosphate into PRPP

PRPP

phosphoribosyl pyrophosphate

required for de novo synthesis

T/F: Pyrimidine bases are built directly of PRPP

FALSE. Purine !!!! Purine bases are built directly of PRPP

purine de novo step 1

Formation of 5-phosphoribosyl-1-amine

Committed step (irreversible)

Glutamine-PRPP amidotransferase combines glutamine with PRPP

Step 10 of purine de novo

Formation of inosine 5'-monophosphate (IMP) aka inosinate

Catalyzed by IMP synthase

IMP structure

hypoxanthine nucleobase + ribose sugar + phosphate

What is the application of inosinate in translation?

Allows one tRNA to recognize multiple codons at the wobble position

What nucleotides can inosinate form hydrogen bonds with?

A, U, C

T/F: IMP is a purine intermediate

True. Branches to be converted either to GMP or AMP

IMP dehydrogenase

IMP -> XMP

then XMP -> GMP

Adenylosuccinate synthetase

IMP -> Adenylosuccinate

-> AMP

GMP kinase

GMP -> GDP

Adenylate Kinase

AMP -> ADP

T/F: synthesis of common nucleotides does not proceed via their free bases

True. PRPP is needed

What does purine de novo synthesis accomplish?

Purine ring precursor hypoxanthine is synthesized stepwise directly on the ribose C-1'

Feedback inhibition in purine de novo

end products inhibit

1. PRPP synthetase

2. glutamine-PRPP amidotransferase

3. branches (adenylosuccinate or IMP dehydrogenase)

Committed step feedback for Purine

AMP, GMP, or IMP inhibit glutamine-PRPP aminotransferase

What does too much GTP promote in the purine de novo pathway?

IMP -> AMP

What does too much ATP promote in the de purine novo pathway?

IMP -> GMP

How is pyrimidine de novo synthesis different from purine de novo?

Pyrimidine synthesis proceeds first by making pyrimidine ring and then attaching to PRPP, rather than attaching base to PRPP first

What is the committed step in pyrimidine de novo?

reaction between aspartate and N-carbamoylphosphate

Aspartate transcarbamoylase (ATCase)

enzyme that catalyzes formation of carbamoyl aspartate in the committed step of pyrimidine de novo synthesis

Steps of pyrimidine de novo synthesis

1. formation of carbomoyl phosphate

2. N-carbamoylasparate

3. dihydroorotate

4. orotate

5. OMP

6. UMP

Carbamoyl phosphate synthetase II

converts glutamine, ATP, and bicarbonate to carbamoyl phosphate in step 1 of pyrimidine de novo

What step forms the pyrimidine ring in pyrimidine de novo synthesis

Step 3: Dihydroorotate

Which step is the redox reaction in pyrimidine de novo synthesis?

Step 4: Orotate

Formation of OMP

Orotate added to PRPP

Step 5 of pyrimidine de novo synthesis

How does OMP convert to UMP?

Loss of CO2 and enzyme orotidylate decarboxylase

UMP kinase

convert UMP -> UDP

nucleoside monophosphate kinase

NDPK

converts UDP -> UTP

nucleoside diphosphate kinases

CTP synthetase

converts UTP -> CTP

Feedback inhibition in pyrimidine de novo

Allosteric inhibition of aspartate transcarbamoylase (committed step) by end-product CTP

How do CTP and ATP affect aspartate transcarbamoylase?

CTP inhibits and ATP restores activity

How is Km affected by CTP?

increases (almost doubles)

How do NDPKs work?

They use ATP as a source of phosphate to convert nucleosides

ATP is usually in excess

Nucleoside monophosphate kinase

UMP + ATP <--> UDP + ADP

Nucleoside diphosphate kinase

XDP + ATP <--> XTP + ADP

Adenylate kinase

AMP + ATP <--> 2ADP

Ribonucleotide reductase

Enzyme converting ribonucleosides to deoxyribonucleosides.

What are the substrates for ribonucleotide reductase

ADP, GDP, CDP, UDP

What kind of reaction is ribonucleotide reductase

Redox to remove the 2'-OH from ribose ring of nucleoside diphosphates

Hydroxyurea

inhibits ribonucleotide reductase

What happens as a result of hydroxyurea inhibiting ribonucleotide reductase?

reduces deoxyribonucleotide pool needed for DNA replication

What diseases is hydroxyurea an agent against?

Cancer like chronic myeloid leukemia and squamous cell cancer of head and neck

dUTP

can replace dTTP in DNA which is bad! comes from dUDP becoming phosphorylated

dUTPase

removes dUTP from DNA pool by removing phosphates and creating dUMP

Why is dUMP converted to DTMP?

to become a methyl group donor

What enzyme converts dUMP -> dTMP?

thymidylate synthase

(and THF)

FdUMP

Suicide inhibitor of thymidylate synthase

designed to look like dUMP but cannot be converted to dTMP due to fluorine on C-5'

Fluorouracil

base in FdUMP

Cofactors of nucleotides

NAD, NADP, FAD

Nicotinamide Adenine Dinucleotide (NAD)

a nucleotide whose synthesis involves Niacin combining with PRPP and ATP

Niacin (Vitamin B3) sources

Diet or Trp

Primary pellagra

deficiency of Trp and Niacin due to diet

Secondary pellagra

deficiency of Trp and Niacin when Niacin is not absorbed properly

Which 2 diseases are examples of secondary pellagra?

Chron's disease and alcoholism

Symptoms of pellagra

dermatitis, dementia, soreness and inflammation of tongue and mouth

NADPH

NAD + phosphate on C-2'

Flavin adenine dinucleotide (FAD)

nucleotide whose synthesis involves Riboflavin

Riboflavin

Vitamin B2

found in liver, almonds, soy nuts, shellfish, milk/dairy, eggs

used to make FAD

Cheilosis

fissuring at corners of mouth caused by deficiency in riboflavin

How are purines degraded?

Dephosphorylation, converting nucleotides to nucleosides and nucleobases

5'-nucleotidase

Removes charge of phosphate groups on purines

Deamination of AMP

AMP -> adenosine -> inosine -> hypoxanthine -> xanthine -> urate

Deamination of GMP

GMP -> guanosine -> guanine -> xanthine -> urate

What is the purpose of xanthine -> urate

This oxidation reaction allows for excretion of uric acid

Salvage pathway

Hypoxanthine -> IMP -> GMP

via recycling and enzyme HGPRT, involves PRPP

HGPRT

Enzyme in purine metabolism that function to salvage purines from degraded DNA to renewed purine synthesis. In this role, it acts as a catalyst in the reaction between guanine and PRPP to form GMP

Diseases from defective purine metabolism

Gout

Lesch-Nyhan Syndrome

Adenosine deaminase (ADA) immunodeficiency

Gout attributed to...

1. hyperactive PRPP synthase. Excess PRPP accelerates purine biosynthesis

2. impaired excretion of uric acid

3. deficiencies in HGPRT, such that hypoxanthine cannot be converted to IMP

T/F: urate has high solubility

False. Urate has low solubility so excess urate leads to precipitations of sodium urate crystals in tissues, causing inflammation and pain

Lesch-Nyhan Syndrome

deficiency of HGPRT

elevated uric acid in blood due to increased de novo purine synthesis

Why is Lesch-Nyhan syndrome thought to occur?

Lack of PRPP consumption by HGPRT increases the availability of PRPP for glutamine-PRPP amidotransferase, enhancing purine de novo synthesis

leads to uric acid production

Symptoms of Lesch-Nyhan syndrome

Biting of fingers, toes, and lips

Gouty arthritis, severe neurological malfunctions

Adenosine deaminase (ADA) immunodeficiency

ADA deficiency causes deoxyadenosine and dATP accumulation. High dATP inhibits ribonucleotide reductase, reducing dNTP production and DNA synthesis

dATP

inhibits ribonucleotide reductase, thereby inhibiting synthesis of dNTPs

leads to immune problems

What immune problems come from ADA immunodeficiency?

T and B lymphocytes do not develop properly