Nucleotides Metabolism

Nucleotides Metabolism Dr ASIFA MAJEED ASSISTANT PROFESSOR DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY ARMY MEDICAL COLLEGE RAWALPINDI

HISTORY Albrecht Kossel (1853-1927), German physiologist and Nobel laureate In1879, Kossel focused his studies on nuclein, a substance found within the nucleus of a cell. Kossel determined that it was composed partly of protein and partly of a nonprotein substance. This second substance consisted of nucleic acids. Nucleic acids consist of nitrogen-bearing compounds known as purines and pyrimidines. From these purines and pyrimidines, Kossel and his colleagues isolated the nitrogen-containing bases cytosine, thymine, adenine, and guanine

Nucleotide Metabolism

Biological Significance of Nucleotide Metabolism • Nucleotides make up nucleic acids (DNA and RNA) • Nucleotide triphosphates are the “energy carriers” in cells (primarily ATP) • Many metabolic pathways are regulated by the level of the individual nucleotides – Example: cAMP regulation of glucose release • Adenine nucleotides are components of many of the coenzymes – Examples: NAD+, NADP+, FAD, FMN, coenzyme A

Nucleotide Metabolism

Medical significance of nucleotide metabolism • Anticancer agents: • Rapidly dividing cells biosynthesize lots of purines and pyrimidines, but other cells reuse them. Cancer cells are rapidly dividing, so inhibitor of nucleotide metabolism kill them • Anti viral agents

Nucleotide Metabolism

Nomenclature Nucleotides are composed of:   

Nitrogenous base Pentose sugar Phosphate groups

Nucleotide Metabolism

Nitrogenous Bases • Aromatic and heterocyclic • Derived from purine or pyrimidine • Numbering of bases is “unprimed”

Nucleotide Metabolism

Important Purines Adenine and guanine are the principal purines of both DNA and RNA.

Adenine

Guanine

Nucleotide Metabolism

Important Pyrimidines Pyrimidines that occur in DNA are cytosine and thymine. Cytosine and uracil are the pyrimidines in RNA.

Uracil

Thymine

Cytosine

Nucleotide Metabolism

Sugars • Pentoses (5-C sugars) • Numbering of sugars is “primed”

Ribose

Deoxyribose

Nucleotide Metabolism

Nucleosides • Result from linking one of the sugars with a purine or pyrimidine base through an N-glycosidic linkage – Purines bond to the C1’ carbon of the sugar at their N9 atoms – Pyrimidines bond to the C1’ carbon of the sugar at their N1 atoms

Nucleotide Metabolism

Nucleosides

Nucleotide Metabolism

Nucleotides • Result from linking one or more phosphates with a nucleoside onto the 5’ end of the molecule through esterification

PYRIMIDINE BIOSYNTHESIS

Pyrimidine is synthesized from carbamoyl phosphate and aspartate

P 1: Carbamoyl phosphate synthesis in the cy

carbamoyl phosphate synthetase II

EP 2: Aspartate transcarbamoylase cataly formation of carbamoylaspartate

committed step

STEP 3: Ring closure

STEP 4: PRPP addition

STEP 5: UTP formation

UMP kinase UMP + ATP

UDP + ADP

Nucleoside diphosphate kinase UDP + ATP UTP + ADP XDP + YTP

XTP + YDP

Nucleoside mono-, di and triphosphates are interconvertable

TEP 6: Amination of UTP results in CTP

CTP synthetase

Hereditary Orotic Aciduria

• an inherited human disease caused by a deficiency in the multifunctional enzyme that catalyzes the last 2 steps in the pyrimidine synthesis • Defect in de novo synthesis of pyrimidines • Loss of functional UMP synthetase – Gene located on chromosome III

• Characterized by excretion of orotic acid • Results in severe anemia and growth retardation • Extremely rare (15 cases worldwide) • Treated by feeding UMP

PURINE BIOSYNTHESIS First purine derivative formed is Inosine Mono-phosphate (IMP) • The purine base is hypoxanthine • AMP and GMP are formed from IMP

PURINE BIOSYNTHESIS De Novo Purine is synthesized from amino acids, tetrahydrofolate and CO2

The committed step in de novo purine synthesis is the activation of PRPP to phosphoribosylamine

C-STEP: PRPP activation

PRPP + Glutamine

Phosphoribosylamine + Glutamate

STEP 1: Addition of glycine

STEP 2: Formylation by N10 -formyltetrahydrofolate

formyltransferase

STEP 3: Transfer of nitrogen from glutamine before ring closure) 8 4

STEP 4: Dehydration and ring closure

-H2O

STEPS 5-8: Carboxylation Aspartate addition Formylation Dehydration and ring closure

STEP 9: Conversion of IMP to ATP and GTP

STEP 9: Conversion of IMP to ATP and GTP

Salvage Pathway for Purines Hypoxanthine or Guanine

Adenine

+ PRPP = IMP or GMP + PPi Hypoxanthineguanosylphosphoribosyl transferase (HGPRTase)

+ PRPP =

AMP + PPi

Adeninephosphoribosyl transferase (APRTase)

Specific Kinases Convert NMP to NDP Nucleoside Monophosphates

Nucleoside Diphosphates

Monophosphate Kinases • Monophosphate kinases are specific for the bases Adenylate Kinase AMP + ATP 2ADP Guanylate Kinase GMP + ATP

GDP + ADP

Lesch-Nyhan syndrome • there is a defect or lack in the HGPRT enzyme • the rate of purine synthesis is increased about 200X • X-linked syndrome • uric acid level rises and there is gout • in addition there are mental aberrations • patients will self-mutilate by biting lips and fingers off

Regulation of purine biosynthesis

CATABOLISM OF PURINES ADENINE + H 2O GUANINE + H 2O

adenase

guanase

HYPOXANTHINE + O

2 + H 2O

XANTHINE + O 2 + H 20

HYPOXANTHINE + AMMONIA XANTHINE + AMMONIA xanthine oxidase xanthine oxidase

XANTHINE + H 2O2 URIC ACID + H 2O2

GOUT • a disorder associated with abnormal amounts of urates in the body • early stage: recurring acute arthritis • late stage: chronic deforming arthritis and eventual renal complication • disease with rich history dating back to ancient Greece • prevails mainly in adult males • symptoms are cause by deposition of crystals of monosodium urate monohydrate

Gout

Therapy of acute gout • treat with colchicine • avoid aspirin • uric acid lowering agents should never be started or stopped during acute attack • pain resolution occurs within 48-72 hrs

Immunodeficiency Diseases Associated with Purine Degradation • Defect in adenosine deaminase – Removes amine from adenosine • SCID- severe combined immunodeficiency • Defect in both B-cells and T-cells (Disease of Lymphocytes) • Patients extremely susceptible to infection

Thymidylate (dTMP) can be synthesized from either CDP or UDP NH2 N O

C

C

N

CH CH

HN

CDP

dCDP

dCTP

ribonucleotide reductase

UDP

dUDP

dUTP H2O

ATP ADP

CH

C CH OO N …-O-P-O-CH 2 O O OH

H2O dCTP deaminase

nucleoside diphosphate kinase

NH3

O C

ATP ADP

PPi

dUMP O

OH HN O

C

C

N

C

CH3

CH

N5,N10 -methyleneTHF thymidylate synthase

dTMP ATP

dTTP

Degradation of Pyrimidines • CMP and UMP degraded to bases similarly to purines – Dephosphorylation – Deamination – Glycosidic bond cleavage

• Uracil reduced in liver, forming β alanine – Converted to malonyl-CoA  fatty acid synthesis for energy metabolism

Regulation of Pyrimidine Biosynthesis • •

Regulation occurs at first step in the pathway () 2ATP + CO2 + Glutamine = carbamoyl phosphate

X Inhibited by UTP

H2N

Enzymes of nucleotide biosynthesis provide targets for cancer chemotherapy

N

O HN O

C

C

NH2

N H

HN C-F CH

FdUMP CO2

C-F

O

CH3 N

C-NH-Glu

Analog of DHF. Inhibits dihydrofolate reductase

thymidylate synthase. OH CO2-

-

+ azaserine H3N C H (O-diazoacetylCH2 L-serine) O - + N=N=CH-C-O-C=O

O

C

N

C CH OO N O-P-O-CH2 O Analog of dUMP. Inhibits O

(in vivo)

5-fluorouracil

methotrexate

N

O

H N

Analog of Gln. Inhibits glutamine amidotransferases (steps 1 & 4 of purine biosynthesis, CTP synthase, & carbamoylphosphate synthetase II).

H3N C H

+

CH2 CH2 H2N-C=O Gln

FORMATION OF DEOXYRIBONUCLEOTIDES • Ribonucleotide reductase studied by Joanne Subbe

• very complex enzyme; contains: • Tyrosine radical • Two catalytically active cysteine residues • Cys are reduced by other proteins – thioredoxin • Ribo. Reductase is the therapeutic target of the anticancer drug hydroxyurea

Ribonucleotide Reductase • The enzyme system consists of 4 proteins – Two of which constitute the Ribonucleotide Reductase (α 2β 2) – Thioredoxin and thioredoxin reductase

• Has three different nucleotide-binding sites – Substrate: NDPs – Activity-determining: ATP & dATP – Specificity-determining: ATP, dTTP, dGTP, and dATP

Conversion of Ribonucleotides to Deoxyribonucleotides 1) A free radical group on the ribonucleotide reductase removes a

hydrogen atom from carbon 3' of ribose and forms a free radical on the ribose of the nucleotide.

2) A thiol group of the enzyme donates a proton to the hydroxy group on carbon 2 followed by the elimination of a molecule of water. 4) Carbon 2 is reduced by the second sufhydryl group. 5) The enzyme donates a hydrogen atom to the free radical (generated in step 1) on carbon 3 to form the deoxyribonucleotide. The enzyme is converted to the original free radical form and must be reduced by thioredoxin to its starting disulfhydryl form.

1 2

3 4

5

deoxyribonucleoside

Conversion of Ribonucleotides to Deoxyribonucleotides

BASE BASE HOCH O H HOCH2 O OH 2 O 5´ 5´ H 1´ H H 1´ 4´ 4´ H H H H 3´ 2´ H 3´ 2´ Ribonucleotide HO OH HO H Reductase Deoxyribonucleoside

Ribonucleoside

Regulation of ribonucleotide reductase The regulation occurs by binding of ribo-NTPs to either the general activity sites or to the specificity sites of the enzyme. The binding of ATP at activity sites leads to increased enzyme activity, while low affinity binding of dATP inhibits the enzyme. To a minor degree dGTP and other dNTP also inhibit ribonuclease. The specificity sites bind ATP, dATP, dGTP, or dTTP with high affinity. The binding of nucleotides at specificity sites effectively allows the enzyme to detect the relative abundance of the four dNTPs and to adjust its affinity for the synthesis of the less abundant dNTPs, to synthesize a balance proportion of dNTP.

NMP to NDP/NTP • Monophosphate kinases are specific for the bases Adenylate Kinase AMP + ATP

2ADP

Guanylate Kinase GMP + ATP

GDP + ADP

UMP kinase UMP + ATP

UDP + ADP

Nucleoside diphosphate kinase UDP + ATP

UTP + ADP

Specific Drug Resistance Methotrexate

• Methotrexate works by inhibiting the function of dihydrolfolate reductase (DHFR) • Cells develop ways to avoid this block – Mutations in DHFR that make it bind less tightly to MTX – Amplication of the DHFR gene (more enzyme activity)

AZT as an Anti-HIV Agent Azido-3’-deoxythymidine Pyrimidine Analogue HIV is a retrovirus RNA genome that is reversetranscribed to DNA • Viral polymerase is inhibited by AZT • • • •

O C

CH3

HN C O C C N H HOCH2 O H H H

H N3

H

READING REFERENCES Lippincott Biochemistry Harper Biochemistry Stryer Biochemistry

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