Nucleic acid metabolism

PBSCTC302 – Intermediary metabolismM.Sc. Biochemistry

Pyrimidines Purines

Structure of purine and pyrimidine bases

Adenine

Hypoxanthine

Guanine

XanthineUracil

Cytosine Thymine

NH3

NH3

NH3 NH3

5’methyl cytosine

CH3

Uric acidO2 O2

Ribose sugar, Nucleoside, Nucleotide, Nucleic acid

⍺-D-ribose sugar ⍺-D-deoxyribose sugar

DeoxynucleotideDeoxynucleoside

nNucleotide triphosphate

H2O 2 Pi

5’ end

3’ end

Chain form

Role of nucleotides

• Cellular concentrations of ribonucleotides are far greater than deoxyribonucleotides. Ribonucleotides not only form RNA but also 2’-deoxyribonucleotides, required for DNA synthesis

• ATP formed by oxidative or substrate level phosphorylation acts as ‘energy-currency’ of cell

• GTP is required for capping mRNA, signal transduction, THB synthesis and microtubule formation

• Nucleotides, such as cAMP and cGMP serve as second messengers in signal transduction

• AMP is a structural component of coenzymes like CoA, FAD, NAD+, and NADP+

• Nucleotides carry activated intermediates in the synthesis of carbohydrates, lipids and conjugated proteins, like UDP-glucose, CDP-choline, SAM, 3-phosphoadenosine 5-phosphosulphate (PAPS)

• During cell cycle, the nucleotide levels are finely regulated by concentrations of key biosynthetic enzymes and their allosteric modulation

• Nucleotides are allosteric effectors for many important steps of intermediary metabolism

Derivatives of Adenine

FAD

cAMP

Cysteine Pantothenate Phosphoadenosine

Coenzyme A

NAD+ NADH

Nucleotide biosynthesis

Purine nucleotides

AMP GMP

GlycineGlutamineAspartate

Pyrimidine nucleotides

Pentose sugars

UMP CMP

Carbon dioxideGlutamineAspartate

John Buchnan

De-novo purine biosynthesis

N10-formyl THFAsp N10-formyl THF

HCO3-

Gln

• In 1950’s, Buchnan used isotopic tracer experiments in birds to determine the origin of N’s of purine ring

Gly

Inosine

Atom position Source

C4, C5 and N7 Glycine

N3 and N9 Glutamine

C2 and C8 C1-THF

C6 HCO3-

N1 Aspartate

Purine nucleus

De-novo purine biosynthesis

5-phosphoribosyl 1-pyrophosphate (PRPP)

5-phospho-β-D-ribosylamine

Glu-PRPP amidotransferase

Gln Glu+ H2O + PPi

1

Glycinamideribonucleotide (GAR)

GAR synthetase

Gly ADP + ATP + Pi

Mg2+

2

Formyl glycinamideribonucleotide (FGAR)

GAR formyltransferase

N10-Formyl THF

3

THF

Formyl glycinamidineribonucleotide (FGAM)

Gln, ATP

Glu, ADP + Pi

FGAM synthetase 4

5-Aminoimidazoleribonucleotide (AIR)

ADP + Pi ATPH2O

AIR synthetase

5

AIR carboxylase

ADP + Pi ATP HCO3

-

Mg2+

Carboxylaminoimidazole

ribonucleotide (CAIR)

6

5-Aminoimidazole-4-(N-succinylcarboxyamide)

ribonucleotide (SAICAR)

SAICAR synthetase

ADP + Pi ATP Asp

7

De-novo purine biosynthesis

Fumarate Adenylosuccinate/ SCAIR lyase

5-aminoimidazole-4-carboxyamide

ribonucleotide (AICAR)

8SAICAR

N10-Formyl THF THF

AICAR transformylase

N-formylaminoimidazole-4-carboxyamide

ribonucleotide (FAICAR)

9

H2O

IMP synthase

Inosinate (IMP)

10

Inosinate (IMP)

Formation of AMP and GMP from IMP branch point

AdenosuccinateGTP + Asp GDP +

P i

Adenylosuccinate

synthetase

Adenylate (AMP)

Adenylosuccinate lyase

Fumarate

NADH + H +

NAD +

Xanthylate

H2 O

IMP dehydrogenase

Guanylate (GMP)

GMP synthetase

ATP AMP + PPi + Gln + Glu+ H2O

Mg2+

De-novo pyrimidine biosynthesisGln Glu + Pi

Carbamic acid

Carbamoylsynthetase II

Pyrimidine

Bicarbonate

ATP ADP

Carboxyphosphate

Carbamoylsynthetase II

Asp

Pi

Carbamoylaspartate

Aspartate transcarbomylase

Dihydroorotate

H2O H+

Dihydroortase

Orotate

NADH + H+ NAD+

Dihydroortatedehydrogenase

ATP ADP

Carbamoyl phosphate

Carbamoylsynthetase II

De-novo pyrimidine biosynthesis

Orotate

Orotidylate

PPi

Orotate phosphoribosyltransferase

Uridylate (UMP)

H+

CO2

Orotidylatedecarboxylase

Cytidylate (CMP)

Glu Gln + ADP ATP+ Pi

Cytidylate synthetase

5-Phosphoribosyl-1-pyrophosphate

Ribose-5-phosphate

PRPP synthetase

ATP AMP

Formation of thymidylate

• The thymine nucleotides are derived from dUMP, which in E.coli is derived from dUTP. In animal cells, dCMP deaminase is induced before DNA synthesis begins for dTMP synthesis via dUMP.

dCMP dUMP

dCMP deaminase

H2O NH3

DHF reductase

THF

Ser

dTMP

N7, N8-DHFN5, N10- Methylene THF

Thymidylate synthase

Gly + H2O

Serine hydroxymethyltransferase

Regulation of de novo purine biosynthesis

5-Phosphoribosyl 1-pyrophosphate

5-Phosphoribosyl amine

FGAR

GAR

FGAM

AIR

CAIR

SCAIR

AICAR

FAICAR

Inosinate

Adenylosuccinate

ADPAMP ATPGDPGTP GMP Xanthylate

• On demand substrate channeling: ‘Purinosome’ complexes comprising enzyme modules are formed when de novo purine synthesis is required by cell

• Negative regulation: Synergistic feedback inhibition of commitment step by nucleotide end-products shuts de novo purine synthesis

• PRPP is a positive regulator. Its consumption shuts de novo purine synthesis

• Rate limiting step: Like purine synthesis, the initial reaction, catalyzed by carbamoyl phosphate synthetase II, is the rate limiting step of the pyrimidine synthesis pathway. However, in E. coli, it is the second reaction, catalyzed by aspartate transcarbomylase, which controls the rate of pathway. This allosteric enzyme has a catalytic and a regulatory domain. While the catalytic domain can act independent of regulatory domain, the presence of regulatory domain senses CTP concentration and decreases the affinity of aspartate binding to catalytic subunit

Regulation of de novo pyrimidine biosynthesis

• Substrate channeling: Carbamoyl synthetase II enzyme has three regions- first responsible for synthesis of carbamic acid, second for release of ammonia from glutamine and third a channel to connect the two. Also, the first three activities of pathway are catalyzed by same 215 kDa protein molecule comprising CPS II, aspartate transcarbomylase and dihydoorotase modules, allowing efficiency by limiting diffusion of intermediates. Similarly, last two activities: orotidylate dehydrogenase and orotidylate pyrophosphorylase are catalyzed by same polypeptide

Catabolism of GMP to uric acid

Guanosine monophosphate

GMP 5’-nucleotidase

Guanosine

H2O PiGuanine

Purine nucleosidephosphorylase (PNP) /

Nucleosidase

H2O + Pi R-5-P

Xanthine

Deaminase

H2O

Pi

Uric acid H2O2 H2O + O2

Xanthine dehydrogenas

e

Xanthine oxidase

NADH + H+ H2O + NAD+

Catabolism of AMP to uric acid

Adenosine monophosphate (AMP) Inosine monophosphate (IMP)

AMP deaminase

H2O NH4+

Adenosine

AMP 5’-nucleotidase

Pi H2O

Inosine

IMP 5’-nucleotidase

H2O Pi

Xantine

H2O + O2 H2O2 + + NAD+ NADH + H+

Xanthine oxidase

Uric acid

Xanthine oxidase

H2O + O2 H2O2 + + NAD+ NADH + H+ Hypoxantine

PNPH2O + Pi

R-5-PR-5-P H2O + Pi

PNP

H2O

Allantoinase

Allantoinate(Some bony fishes)

Catabolism of uric acid to ammonia

Uric acid(Primates, birds, reptiles, insects)

Allantoin(Most mammals;turtles; some insects; gastropods)

½ O2 + H2O CO2

Urate oxidase

Excreted by:

4 NH3

Ammonia(Plants; crustaceans; many marine vertebrates)

Urease

2 CO2 2 H2O

2 H2O

2 Urea(Amphibians, cartilaginous fishes, marine vertebrates)

Glycolate

Allantoicase

+

Catabolism of pyrimidines

H2O

NH3

Cytosine

Uracil

Thymine

H2O

H2O

Carbamoyl-β-alanine

Carbamoyl-β-aminoisobutyrate

Dihyropyrimidinase

Dihyropyrimidinase

NH3

⍺-Ketoglu Glu

Methylmalonylsemialdehyde

Malonate

Aminotransferase

Aminotransferase

NH4+

+ HCO3-

NH4+

+ HCO3-

Ureidopropionase

Ureidopropionase

β-aminoisobutyrate

β-alanine

NADPH + H+ NADP+

NADPH + H+ NADP+

Dihyrouracildehydrogenase

Dihyrouracildehydrogenase

Dihyrouracil

Dihyrothymine

Adenine GuanineHypoxanthine

Purine salvage

Inosinate (IMP) Guanylate (GMP)

HGPRT

PRPP

PPi

Adenosine

PRPP

PPi

APRT

Adenylate (AMP)

Inosine Guanosine

Syndromes or diseases due to defects in degradation of purine nucleotides

1. GOUT- Gout is a common condition due to high blood and tissue concentrations of

uric acid caused by deregulation of de novo purine biosynthesis. In gout, precipitation

of sodium urate in kidneys and regions of body with temperature below 37 , like ℃joints and extremities results in complications in renal handling and inflamed, painful

and arthritic joints. A combinatorial therapy involves taking diet low in nucleotides

(avoiding red meat, beer and dried beans) and taking drugs such as allopurinol (a

hypoxanthine analog that acts as suicide inhibitor of xanthine oxidase), anticancer and

antihyperuricemic drugs. Gout may also result from faulty carbohydrate metabolism,

wherein deficiency of glucose-6-phosphatase (von Gierke’s disease) results in

accumulation of ribose-5-phosphate (R-5-P) instead of glucose. R-5-P leads to excess

5-Phosphoribosyl-1-pyrophosphate (PRPP) which stimulates purine synthesis, thus

producing more uric acid. Gout is more common in men. In women, oestrogen

promotes uric acid excretion

Swollen and inflammed

joints

Uric acid crystals

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2. LESCH NYHAN SYNDROME (LNS) – An X-linked recessive genetic disease caused due to mutations in HGPRTase

gene resulting in severe deficiency or complete lack in activity of HGPRTase (hypoxanthine guanine

phosphoribosyl transferase) which salvages guanine and hypoxanthine. If de novo pathway is dysfunctional,

AMP can be converted to GMP via IMP by APRTase (adenine phosphoribosyl transferase). In LNS, rather than

being salvaged, A and G are broken down, leading to excess uric acid. Patients excrete 4-5 times as much uric

acid as gout patients do. Besides, neurological problems like spasticity, mental retardation and self mutilation

ensue, due to imbalanced purine nucleotide concentrations during CNS development

3. Immunodeficiency diseases (SCID-SEVERE COMBINED IMMUNODEFICIENCY DISEASES) – SCID is due to

defects in purine nucleoside degradation due to a range of genetic mutations in enzymes of purine

catabolism and salvage pathways. Adenosine deaminase (ADA) and Purine nucleoside phosphorylase

(PNP) deficiency causes SCID. It is also called as the bubble boy disease due to lack of immune protection

and neurological defects

Syndromes or diseases due to defects in degradation of purine nucleotides

Pyrimidine salvage

Cytidine deaminase

H2O

NH3

Zn2+

Uridine

Cytidine

Gln + ATP

Glu+ ADP + Pi

CTP synthase

Cytidine diphosphate (CDP)

Cytidylate kinase

Nucleotide diphosphatephosphatase

ATP ADP

Pi H2O

UDP

ATP ADP

Pi H2O

Mg2+

Ca2+

Cytidine kinase

5’ nucleotidase

Cytidylate (CMP)

ATP ADP

ATP ADP

Pi H2O

Mg2+

Ca2+

UMP

Pi H2O

Cytidine triphosphate (CTP)

ATP ADP

Pi H2O

Nucleoside triphosphatephosphatase

UTP

ATP ADP

H2O Pi

Mg2+

Ca2+

Nucleoside diphosphate

kinase

Ca2+Apyrase

PPi 2H2O

Formation of deoxy derivatives of nucleotides

Ribonucleotide reductase

Thioredoxin reductase

• Ribonucleotide diphosphates are converted to 2’ deoxy-ribonucleotides by ribonucleotide diphosphate reductase (RDR), an enzyme complex, comprising two B1 and two B2 subunits. It is active only in dividing

cells. It is subject to complex allosteric control by nucleotide triphosphates. The reaction requires a small protein thioredoxin with two free sulfhydryl groups positioned in such a way as to form a disulphide bond. Another enzyme, thioredoxin reductase regenerates reduced thioredoxin using FADH2 and NADPH.

ThioredoxinSHSH

ThioredoxinSS

NADPH + H+NADP+

Ribonucleoside diphosphate(ADP, GDP, CDP, UDP)

2’ deoxy-ribonucleoside diphosphate(dADP, dGDP, dCDP, dUDP)

 Formation of nucleoside di and  tri-phosphates

• Nucleoside monophosphates are converted to their di- and tri-phosphate derivatives by phosphorylation reactions catalyzed by nucleoside monophosphate kinases (NMP) and nucleoside diphosphate kinases (NDP) using ATP.

Nucleoside monophosphate

Nucleoside diphosphate

Nucleoside diphosphate

ATP

ADP

ATP

ADP

NMP kinase

NDP kinase

Resources• Principles of Biochemistry by Horton, Moran, Scrimgeour, Perry and

Rawn• Biochemistry: A case oriented approach by Montgomery, Conway,

Spector and Chappell• Biochemistry by Jeremy M. Berg, John L. Tymoczko and Lubert Stryer