Metabolism of Nucleotides

Guided by

Dr. D V Bhale

( prof. & HOD)

By

Roshan Kumar Mahat

(PG Student)

Department Of Biochemistry

Objectives to know

1. Nitrogen bases i.e. purines & pyrimidines

2. Nucleosides

3. Nucleotides

4. Synthesis of purine nucleotides

5. Regulation of purine nucleotides synthesis

6. Inhibitors of purine nucleotide synthesis

7. Disorders of purine metabolism

8. Synthesis of pyrimidine nucleotides

9. Inhibitors of pyrimidine nucleotides synthesis

10. Disorders of pyrimidine nucleotide synthesis

Pyrimidines and PurinesPyrimidine and purine are the names of the

parent compounds of two types of nitrogen-containing heterocyclic aromatic compounds.

N

N

N

N

N

NH

Pyrimidine Purine

Important Pyrimidines• Pyrimidines that occur in DNA are cytosine

and thymine. Cytosine and uracil are the pyrimidines in RNA. HN

NH

O

O

Uracil

HN

NH

O

O

CH3

Thymine

HN

NH

NH2

O

Cytosine

Important Purines• Adenine and guanine are the principal

purines of both DNA and RNA.

Adenine

N

N

NH2

N

NH

Guanine

O

HN

NH

N

N

H2N

Caffeine and Theobromine• Caffeine (coffee) and theobromine (coffee

and tea) are naturally occurring purines. Caffeine

N

N

O

N

N

H3C

O

CH3

CH3

Theobromine

O

HN

NN

N

CH3

CH3

O

Nucleosides

• Is a structure formed by the combination of nitrogen base and sugar.

N2 base Sugars Nucleoside

Adenine Deoxyribose/Ribose Adenosine

Guanine Deoxiribose/Ribose Guanosine

Thymine Deoxyribose Thymidine

Cytosine Deoxyribose/Ribose Cytidine

Uracil Ribose Uridine

Nucleotides

• Nucleotides are phosphoric acid esters of nucleosides.

Nucleoside Phosphoric acid Nucleotides

Adenosine Phosphoric acid Adenylate (AMP)

Guanosine Phosphoric acid Guanylate ( GMP)

Thymidine Phosphoric acid Thymidylate ( TMP)

Cytidine Phosphoric acid Cytidylate (CMP)

Uridine Phosphoric acid Uridylate (UMP)

Synthesis of purine nucleotides

Synthesis of purine nucleotidesDenovo synthesis

Synthesis of purine base step by step on the ribose 5-phosphate

Salvage pathwayAddition of ribose 5-phosphate to the preformed purine bases or

addition of phosphate to the purine nucleosides

Sources of different atoms of purine ring

Denovo synthesis of purine nucleotides

Tissue and site of synthesis

Tissues- major tissue is liver

Site- cytosol

Inhibitors

1. Sunfonamide Are structural analogues of PABA Act as competitive inhibitors of synthesis of folic

acid from PABA in bacteria. They inhibit the reactions of purine nucleotide

synthesis requiring folic acid ( GAR transformylase and AICAR transformylase)

Used as bacteriostatic drugs to control bacterial infection.

2. Methotrexate and AminopterinAre structural analogue of folic acid.They act as a competitive inhibitors of

dihydrofolate reductase thus blocking the biosynthesis of tetrahydrofolic acid.

They inhibit the reaction requiring folic acid for purine nucleotide synthesis.

Used in t/t of cancers like leukemia choriocarcinoma.

3. Trimethoprim structural analogue of folic acid.Acts as a competitive inhibitors of

dihydrofolate reductase in bacteria thus blocking the biosynthesis of tetrahydrofolic acid.

Inhibit the reaction requiring folic acid to purine nucleotide synthesis.

Used in the t/t of bacterial infections and UTI.

4. 6-mercaptopurine is a structural analogue of purine bases. is converted to 6- thioionosine

monophosphate by the enzyme HGPRT, called lethal synthesis.

6-thio IMP inhibits the conversion of IMP to AMP and GMP.

6-thio IMP also feed back inhibits glutamine PRPP amidotransferase.

Used as an anticancer drug.

5. Thioguanine Is a guanine analogue. It is converted to 6-thio GMP by the

enzyme HGPRT. 6-thio GMP inhibits the conversion of IMP

to GMP. Also inhibits glutamine PRPP

amidotransferase. Used as an anticancer drug.

6. Azaserine is a structural analogue of glutamine. is a glutamine antagonist. inhibits the enzyme reactions in purine and

pyrimidine nucleotide synthesis that utilize glutamine as a substrste.

it is highly toxic to the cells so it is not used clinically as a drug.

Regulation

1. Intracellular conc. Of PRPP-

depends upon 2 factors i.e. its synthesis & utilization.

Synthesis depends on- Availability of R-5-P. Action of enzyme PRPP synthetase.

Utilization depends on- Denovo synthesis. Salvage pathway.

2. Activity of enzyme PRPP amidotransferase.Increased activity of PRPP

amidotransferase leads to Increased synthesis of AMP amd GMP, which feedbackly inhibit the enzyme PRPP amidotransferase.

3. Both AMP and GMP inhibit their own formation by feedback inhibition of adenylosuccinate synthetase and IMP dehydrogenase.

Salvage pathway

It refers to the formation of purine nucleotides by the

1. Addition of ribose phosphate ( from PRPP) to the preformed purine bases.

2. Addition of phosphate to the preformed purine nucleosides.

Significance

Salvage pathway provide a pathway for the utilization of purine bases derived from diet (exogenous) and normal turnover of the nucleic acids.

In erythrocytes, denovo syntheis of purine nucleotides does not occur because of absence of PRPP amidotransferase. The requirement of purine nucleotides is met by the salvage pathway.

Synthesis of purine nucleotides from purine bases

Catalyzed by HGPRT and APRT.

Adenine + PRPP AMP + PPi

Hypoxanthine + PRPP IMP + PPi

Guanine + PRPP GMP+ PPi

APRT

HGPRT

HGPRT

Synthesis of purine nucleotides from purine nucleosides

Adenosine + ATP AMP+ ADP Adenosine kinase

Degradation of purine nucleotides

Disorders of purine metabolism

1. Gout

2. Lesch nyhan syndrome

3. Immunodeficiency associated with purine metabolism

4. Infantile autism

Gout

Metabolic disorders associated with overproduction of uric acid.

At physiological form, uric acid is found in more soluble form as sodium urate.

In severe hyperuricemia, crystal of sodium urate get deposited in the soft tissues, particularly in joints. Such deposits are commonly known as tophi.

This causes inflammation of joints resulting in gouty arthritis.

The prevalence of gout is about 3 per 1000 persons, mostly affecting males.

Post menopausal women, however are as susceptable as men for this disease.

Historically, gout was found to be associated with high living, over eating and alcohol consumption.

Lead poisoning also causes gout by decreasing uric acid excretion.

Types of gout

Gout

Primary gout

Metabolic gout

Renal gout

Secondary gout

Metabolic gout

Renal gout

Primary metabolic goutIt is an inborn error of purine metabolism

due to overproduction of uric acid.

Causes:

1. Increased activity of PRPP synthetase

2. Overactivity of PRPP amidotransferase

3. HGPRT deficiency

4. Glucose 6-phosphatase deficiency

5. Elevation of glutathione reductase

Primary renal gout

It is due to failure of uric acid excretion from the body so that uric acid level in the body gets increased.

Secondary metabolic gout

Secondary gout is due to secondary to certain diseases like leukemia, polycythemia, lymphoma, psoriasis and increased tissue breakdown like in trauma, starvation etc.

Secondary renal gout

It is due to secondary to defective glomerular filtration of urate due to generalized renal failure.

Tratment of gout

Is by

1. Use of colchicine & uricosuric drugs. To remove urates from the joint, colchine

is the drug of choice. To remove the urates from the body,

urocosuric drugs such as probenecid, sulfinpyrazole, salicylates etc are used.

2. Use of allopurinol-inhibits the activity of enzyme xanthine oxidase as a result of which uric acid is not produced.

Lesch Nyhan syndromeFist described tn 1964 by Michael Lesch( a

medical student) and William L. Nyhan (his teacher).

It is X linked metabolic disorder since the structural genes for HGPRT is located on the X chromosome.

It affects only males and is characterized by excessive uric acid production and neurological abnormalities such as mental retardation, aggressive behaviour, learning disability etc.

The patients of this disorder have an irresistible urge to bite their fingers and lips,ofen causing self-mutilation.

Biochemical basisHGPRT deficiency spares the utilization of

PRPP through salvage and the accumulated PRPP takes part in the purine biosynthesis by the denovo pathway finally leading to hyperuricemia.

The biochemical basis for neurological abnormalities are big enegma till date.

Indeed, it is surprising that the deficiency of a single enzyme can cause such an abnormal behavioural changes.

few explanations are putforth in this regard.

Neurological symptoms may be due to decreased availability of purines to the developing brain which has a low capacity for denovo purine synthesis and hence depends on purine salvage pathway for the supply of purine nucleotides it requires.

Treatment

allopurinol is used to treat hyperuricemia but it has no effect on the neurological menefestation in theses patients.

Treatment for the neuro-behavioural features are limited to behavioural therapy and providing protective physical device to prevet self-mutilation.

Immunodeficiency diseases associated with purine metabolism

Two different immunodeficiency disorders associated with degredation of purine nucleotides are known.

The enzyme defects are adenosine deaminase and purine nucleoside phosphorylase, involved in uric acid synthesis.

The deficiency of ADA causes SCID involving T- cell and usually B- cell dysfunction.

It is explained that ADA deficiency results in the accumulation of Datp which is an inhibitor of ribonucleotide reductase and thus DNA synthesis, replication are adversely affected.

Different modes of t/t such as blood transfusion, bone marrow transplantation are tried to cure the diseases but with limited effects.

But, like in any other inborn error, the real hope for the future is only gene therapy.

In 1990, a 5 year old girl suffering from SCID was successfully cured by transfecting the ADA gene into stem cells of the patients.

This is considered as landmark in the history of trating inborn errors of metabolism.

The deficiency of purine nucleoside phosphorylase is associated with impairement of T cell function but has no effect on B cell function.

It is believed that d GTP inhibits the development of normal T-cells.

Infantile Autism

Recently it was observed that children suffering from infantile autism exihibited increased excretion of uric acid but surprisingly the serum concentrations are within normal limits.

The biochemical basis for this is unknown.An oral dose of uridine is tried in the t/t.

Synthesis of pyrimidine nucleotides

Synthesis of pyrimidine nucleotides

Denovo synthesisSynthesis of pyrimidine nucleotide

refers to the formation of pyrimidine ring structure followed

by the addition of ribose phosphate.

Salvage pathwayFormation of pyrimidine

nucleotides from pyrimidine bases

Sources of different atoms of prrimidine rings

Denovo synthesis of pyrimidine nucleotides

Tissue and site of synthesisMainly occurs in the liver.The reaction occurs in cytosol and

mitochondria. The formation of orotate from dihydroorotate occurs ie mitochondria and all other reactions occur in the cytosol.

2 ATP + HCO3- + Glutamine + H2O

CO

O PO3-2

NH2

Carbamoyl Phosphate

NH2

CNH

CH

CH2

C

COOO

HO

O

Carbamoyl Aspartate

HN

CNH

CH

CH2

C

COOO

O

Dihydroorotate

HN

CNH

C

CHC

COOO

O

Orotate

HN

CN

C

CHC

COOO

O

HH

CH2

OH OH

H HO

O2-O3P

Orotidine-5'-monophosphate(OMP)

HN

CN

CH

CHC

O

O

HH

CH2

OH OH

H HO

O2-O3P

Uridine Monophosphate(UMP)

2 ADP +Glutamate + Pi

CarbamoylPhosphateSynthetase II

AspartateTranscarbamoylase(ATCase)

Aspartate

Pi

H2O

Dihydroorotase

Quinone

ReducedQuinone

DihydroorotateDehydrogenase

PRPP PPi

Orotate PhosphoribosylTransferase

CO2

OMP Decarboxylase

Pyrimidine Synthesis

UMP UTP and CTP

• Nucleoside monophosphate kinase catalyzes transfer of Pi to UMP to form UDP; nucleoside diphosphate kinase catalyzes transfer of Pi from ATP to UDP to form UTP

• CTP formed from UTP via CTP Synthetase driven by ATP hydrolysis – Glutamine provides amide nitrogen for C4 in

animals

UDP dUDP

dUDP dUMP

dUMP dTMP

N5,10 formyl THF formyl THF

Ribonucleotide reductase

Thymidylate synthetase

Regulation of pyrimidine synthesis

In bacteria, aspartate transcarbamoylase catalyses a committed step in pyrimidine biosynthesis.

Aspartate transcarbamoylase is a good example of an enzyme controlled by feedback mechanism by the end product CTP.

In certain bacteria, UTP also inhibits aspartate transcarbamoylase. ATP, however stimulates aspartate transcarbamoylase activity.

Carbamoyl phosphate synthase II is the regulatory enzyme of pyrimidine synthesis in animals.

It is activated by PRPP and ATP and inhibited by UDP and UTP.

OMP decarboxylase inhibited by UMP and CMP, also controls pyrimidine formation.

Inhibitors of pyrimidine synthesis

SulfonamidesMethotrexateTrimethoprim5-fluorouracilFluorocytosine

Salvage pathwaySalvage pathway of pyrimidine nucleotide

synthesis refers to the formation of pyrimidine nucleotides from pyrimidine bases.

Significance Salvage pathway provide a pathway for the

utilization of pyrimidine bases derived from diet(exogenous) and normal turnover of nucleic acids.

Enzymes and reactions

There are 2 enzymes that catalyze the reactions of salvage pathway. They are uracil phosphoribosyl transferase (UPRT) and thymidine kinase.

Uracil + PRPP UMP + PPi

Thymidine + ATP TMP+ ADP

UPRT

Thymidine kinase

Disorders of pyrimidine metabolism

Disorders of pyrimidine metabolism includes:Orotic aciduriaReye’s syndrome

Orotic aciduriaIs a rare metabolic disorder characterized by the

excretion of orotic acid in urine, severe anemia and retarded growth.

It is due to the deficiency of the enzymes orotate phosphoribosyl transferase and OMP decarboxylase of pyrimidine synthesis.

Both these enzymes activities are present on a single protein as domains (bifunctional enzyme).

Treatment Feeding diet rich in uridine or cytidine is an

effective t/t of orotic aciduria.These compounds provide pyrimidine

nucleotides required for DNA and RNA synthesis.

Reye’s syndrome

Is considered as a secondary orotic aciduria.It is believed that a defect in ornithine

transcarbamoylase (of urea cycle) causes the accumulation of carbamoyl phosphate.

This is then diverted for the increased synthesis and excretion of orotic acid.

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