Topic 13: Biosynthesis & Degradation of Nucleotides

multiple roles of Nucleotides in the cell (why are they essential molecules?)

1. Genetic material precursors → RNA and DNA

2. Components of coenzymes → NAD⁺, NADP⁺, FAD, FMN, SAM

3. Activated intermediates → e.g., UDP-glucose in glycogen synthesis

4. Second messengers → cAMP, cGMP

5. Energy carriers → ATP, GT

Metabolic significance of nucleotides

- Most cells can synthesize nucleotides → highlights their importance

- Not a major energy source

- Degradation products are recycled (salvage pathways) or excreted (not oxidized for energy)

What are Nucleotides central to?

information storage, energy transfer, and cellular regulation, rather than energy production

structure of Nucleotides - 3 components

- Nitrogenous base

- Sugar

- Phosphate group

Types of nucleotide bases

- Purines: Adenine (A), Guanine (G)

- Pyrimidines: Cytosine (C), Uracil (U), Thymine (T)

- RNA: A, G, C, U

- DNA: A, G, C, T

nucleotide Sugars

RNA: Ribose

DNA: Deoxyribose

Key structural bonds of nucleotides

- Base + sugar → Nucleoside (via β-N-glycosidic bond)

- Purine → N9 to C1′

- Pyrimidine → N1 to C1′

- Nucleoside + phosphate → Nucleotide (phosphate at C5′)

Two pathways to make nucleotides (biosynthesis)

1. De novo pathway: Builds purine & pyrimidine rings from small molecules. Precursors: CO₂, NH₃, amino acids

2. Salvage pathway: Recycles free bases from DNA/RNA breakdown

Key molecules - nucleotide biosynthesis

- PRPP (5-phosphoribosyl-1-pyrophosphate): → Ribose donor for both purine & pyrimidine synthesis

- N¹⁰-formyltetrahydrofolate: → Provides formate groups in purine synthesis

- In purine synthesis: C2 and C8 of the purine ring come from N¹⁰-formyl-THF

way nucleotides are synthesized?

continuously synthesized (low cellular pool)

What is nucleotide biosynthesis essential for?

Essential for DNA & RNA synthesis → cell division

result of inhibiting nucelotide biosynthesis

→ blocks cell growth → Target of many cancer drugs

How do Cells maintain nucleotide supply?

through de novo synthesis + recycling (salvage) to support rapid DNA/RNA production

De novo Purine Synthesis - Overview

- Major site: Liver

- Ribose donor: PRPP (from ribose-5-phosphate + ATP)

- PRPP synthesis enzyme: PRPP synthetase → Inhibited by ADP, GDP

- Purine ring is built directly on ribose

- Structure is assembled step-by-step on PRPP

Committed Step & Regulation

- Committed step: PRPP + glutamine → 5-phosphoribosylamine (enzyme: glutamine-PRPP amidotransferase)

- This is the main control point of purine synthesis

- Introduces N9 nitrogen of purine ring

- Inhibited by: AMP, GMP, IMP (feedback inhibition)

Building the Purine Ring

- Atoms come from Glutamine, glycine, aspartate, CO₂, N¹⁰-formyl-THF → C2 & C8

- Folate (Vitamin B9) is the is the precursor for N¹⁰-formyl-THF

- Folate deficiency → ↓ purine synthesis

IMP (inosine monophosphate)

- = central branch point

- the precursor for both AMP and GMP

- 1st complete purine nucleotide

Formation of IMP (Branch Point)

- nitrogenous base attached to the sugar is called hypoxanthine

AMP synthesis

- IMP + aspartate → adenylosuccinate → AMP + fumarate

- Requires GTP

- Inhibited by AMP

GMP synthesis

- IMP → XMP (requires NAD⁺) → GMP

- Uses glutamine

- Inhibited by GMP

- AMP uses GTP, GMP uses ATP → balanced production

Organization of the Pathway - formation of AMP & GMP

- Bacteria: separate enzymes

- Eukaryotes: multifunctional enzyme complexes

- To go from PRPP → IMP, This pathway involves ~10-11 steps

- Enzymes often form complexes for efficiency

Regulation of Purine Biosynthesis (E. coli) - Committed step regulation

- Glutamine-PRPP amidotransferase is inhibited by IMP, AMP, and GMP •

- AMP and GMP show synergistic inhibition (combined effect > individual effects)

Regulation of Purine Biosynthesis (E. coli) - Branch point step regulation (IMP → AMP or GMP)

- IMP → XMP → GMP pathway inhibited by GMP

- IMP → adenylosuccinate → AMP pathway inhibited by AMP

Regulation of Purine Biosynthesis (E. coli) - Outcome

- Each nucleotide (AMP or GMP) inhibits its own synthesis specifically

Results when AMP and GMP levels are high

→ IMP accumulates → Further inhibits glutamine-PRPP amidotransferase (global shutdown)

Energy balance mechanism - Balancing Nucleotide Pools

- AMP synthesis requires GTP

- GMP synthesis requires ATP → Ensures balanced production of purines

PRPP synthesis regulation - Balancing Nucleotide Pools

PRPP synthetase inhibited by ADP and GDP

Overal concept - Balancing Nucleotide Pools

Purine biosynthesis is tightly regulated by feedback inhibition and energy balance to maintain equal levels of AMP and GMP

Key difference of pyrimidine vs. purines

Pyrimidine ring is synthesized first, then attached to ribose (PRPP)

De novo Synthesis of Pyrimidine Nucleotide (ENTIRE PROCESS)

- Major precursors: Carbamoyl phosphate + Aspartate + PRPP

- Formation of carbamoyl phosphate: CO₂ + NH₄⁺ (from glutamine) + ATP Enzyme: Carbamoyl phosphate synthetase II (cytosol)

- Committed step: Carbamoyl phosphate + aspartate → N-carbamoyl aspartate Enzyme: Aspartate transcarbamoylase

- Ring formation steps: N-carbamoyl aspartate → dihydroorotate → orotate

- Attachment to ribose: Orotate + PRPP → orotodylate (OMP)

- Final products: OMP → UMP → UTP → CTP

Regulation of pyrimidine nucleotide synthesis

- mostly at the step catalyzed by aspartate transcarbomylase (ATCase)

- The bacterial enzyme consists of six catalytic subunits and six regulatory subunits

- CTP binds regulatory subunit and inhibits the enzyme due to conformational changes caused by CTP

- Maximum enzyme activity of the enzyme is obtained in the absence of CTP

- ATP prevents inhibition by CT

Synthesis of NDPs and NTPs

- NDPs and NTPs are formed by stepwise (NMP → NDP → NTP)

- Phosphorylation of nucleotides using specific kinases

Synthesis of NDPs and NTPs - STEPS

1. ATP + AMP --> 2 ADP : Catalyzed by adenylate kinase

2. ADP --> ATP : Glycolysis, oxidative phosphorylation etc

3. ATP + NMP--> ADP + NDP : Catalyzed by nucleoside monophosphate kinase (specific to base, nonspecific to sugar)

4. NTPD + NDPA --> NDPD + NTPA Catalyzed by nucleoside diphosphate kinase (nonspecific to base, nonspecific to sugar)

Synthesis of deoxyribonucleotides - ENZYME & SUBSTRATE

- Enzyme: ribonucleotide reductase

- Substrate: NDPs

Synthesis of deoxyribonucleotides - PROCESS

- Substrate is reduced by donating electrons from 2 -SH groups on the enzyme

- 2 -SH groups oxidized to disulfide bond during the rxn

- Regeneration of reduced form of enzyme achieved by two pathways in which the ultimate electron donor of both is NADPH

- enzyme is regulated to achieve balanced synthesis of dNTPs

ribonucleotide reductase

- In E. coli and most eukaryotes, the enzyme is a dimmer of two subunits, R1 (or α) and R2 (or β)

- Catalytic site is formed by R1 and R2

- 2 allosteric sites in R1 subunits

Regulation of dNTP synthesis by ribonucleotide reductase

1. One allosteric site can bind ATP and dATP and controls overall activity

- ATP increases overall activity and dATP inhibits overall activity. Small amount of dATP stimulates the activity

2. Other allosteric site can bind ATP, dATP, dGTP and dTTP and controls substrate specificity

- ATP or dATP changes the specificity of the enzyme to CDP and UDP

- dTTP changes the specificity of the enzyme to GDP

- dGTP changes the specificity of the enzyme to ADP

Ribonucleotide Reductase - Function

- Converts NDP → dNDP (essential for DNA synthesis) by removing the -OH at C2′ and replace it with H

- ONLY difference: RNA has -OH at C2′ DNA has H at C2′

Ribonucleotide Reductase R2 subunit

- tyrosyl free radical located in the R2 subunit

- The tyrosyl radical cannot act directly → generates a secondary radical at the active site (R1 subunit), passing the spark to where the reaction happens

tyrosyl free radical

- tyrosine amino acid that has lost one electron, creating an unpaired electron

- like a spark plug

Ribonucleotide Reductase Mechanism (dNTP Synthesis) - 6 STEPS

1. Radical Initiation: A radical is generated and transferred to the active site & Radical forms at C3′ of ribose

2. Removal of 2′-OH: C3′ radical facilitates removal of -OH at C2′ and -OH leaves as H₂O

3. Intermediate Formation: Formation of a carbonium ion (carbocation) at C2′ which is a high-energy intermediate

4. Stabilization: C3′ radical stabilizes the carbocation intermediate & ensures controlled rxn progression

5. Reduction: Electrons supplied by -SH groups of two cysteine residues (R1 subunit) and converts C2′ into C-H (deoxy form)

6. Radical Reset: Radical is transferred back → enzyme returns to original state

radical-driven reaction

removes the 2′-OH and converts RNA building blocks into DNA building block

Ribonucleotide Reductase Mechanism (dNTP Synthesis) - OUTCOME

- Ribonucleotides → Deoxyribonucleotides (dNDPs)

- Essential for DNA synthesis

Ribonucleotide Reductase Mechanism (dNTP Synthesis) - KEY CONCEPT

- A long-range radical transfer mechanism enables precise removal of the 2′-OH

- Ensures accurate formation of deoxyribose

Thymine synthesis (dTMP)

- not synthesized de novo because pyrimidine biosynthesis produces uracil (UMP)

- generated by methylation of dUMP via thymidylate synthase, allowing efficient regulation and coupling to folate metabolism

Synthesis of Thymidylate (dTMP) - REACTION

dUMP → dTMP (methylation reactioN)

Synthesis of Thymidylate (dTMP) - ENZYME

Thymidylate synthase

Synthesis of Thymidylate (dTMP) - WHERE DOES DUMP COME FROM?

- Two main routes

1. From dCTP (deamination)

2. From dUTP (via dUTPase → dUDP → dUMP) •

- dUTP must be converted → prevents uracil entering DNA

Synthesis of Thymidylate (dTMP) - METHYL DONOR

N5,N10-methylenetetrahydrofolate (THF derivative)

Synthesis of Thymidylate (dTMP) - Where does the methyl group come from?

- Donor: N⁵,N¹⁰-methylene-THF (a folate derivative)

- Role: Supplies 1-carbon unit → becomes CH

Synthesis of Thymidylate (dTMP) - What happens to THF?

- During the reaction: THF → becomes DHF (dihydrofolate)

- It gets used up

Synthesis of Thymidylate (dTMP) - Regeneration

- DHF → THF

- enzyme: Dihydrofolate reductase (DHFR)

- eses: NADPH

- Recycles THF so the reaction can continue

Synthesis of Thymidylate (dTMP) - Clinical Relevace

- If you block DHFR: No THF regeneration, No dTMP, No DNA synthesis

- That's how some anticancer drugs work

Nucleotide Salvage Pathway - Purpose

- Recycles free bases from nucleic acid degradation → energy-efficient pathway

- faster and energy-saving compared to de novo synthesis

Nucleotide Salvage Pathway - Purine Salvage

- Adenine + PRPP → AMP + PPi (enzyme: adenosine phosphoribosyltransferase)

- Hypoxanthine + PRPP → IMP + PPi

- Guanine + PRPP → GMP + PPi (enzyme: HGPRT)

Nucleotide Salvage Pathway - Pyrimidine Salvage

- Uracil → UMP (via uridine intermediate) (uridine phosphorylase, uridine kinase)

- Thymine → dTMP (via thymidine intermediate) (thymidine phosphorylase, thymidine kinase)

Nucleotide Salvage Pathway - Key Molecule

PRPP provides ribose-phosphate for nucleotide formation

Chemotherapy & Nucleotide Biosynthesis Inhibition - Key drug mechanisms

1. Methotrexate & Aminopterin

2. Acivicin (glutamine analog)

3. Fluorouracil (5-FU)

- they block DNA synthesis → prevent cancer cell proliferation

chemotherapy & Nucleotide Biosynthesis Inhibition - Why target this pathway?

Cancer cells divide rapidly → need high nucleotide supply for DNA synthesi

chemotherapy & Nucleotide Biosynthesis Inhibition - Methotrexate & Aminopterin Mechanism

Folic acid analogs inhibit dihydrofolate reductase (DHFR). ↓ dTMP → ↓ dTTP → blocks DNA synthesis

chemotherapy & Nucleotide Biosynthesis Inhibition - Acivicin (glutamine analog) Mechanism

Inhibits glutamine amidotransferases & blocks nitrogen donation in nucleotide synthesis

chemotherapy & Nucleotide Biosynthesis Inhibition -Fluorouracil (5-FU) Mechanism

- Target: Thymidylate synthase

- dUMP → dTMP (adds methyl group) •

- 5-FU is converted into FdUMP inside cells

- Which mimics dUMP and irreversibly inhibits thymidylate synthase, blocking DNA synthesis

- Acts as a suicide inhibitor (irreversibly binds enzyme)

Purine Nucleotide Degradation - END PRODUCT IN HUMANS

Uric acid (produced in liver, excreted in urine)

Purine Nucleotide Degradation - PATHWAY OVERVIEW

1. AMP & GMP → nucleosides (via nucleotidases)

2. Guanosine → guanine → xanthine (guanine deaminase)

3. Adenosine → inosine → hypoxanthine (adenosine deaminase + nucleosidase)

4. Hypoxanthine → xanthine → uric acid (xanthine oxidase)

Purine Nucleotide Degradation - KEY ENZYME

Xanthine oxidase (final steps)

Purine Nucleotide Degradation - IMPORTANT NOTES

- Humans excrete ~0.6 g urate/day

- Other mammals → uric acid → allantoin

- Some organisms → final product = ammonia

Pyrimidine Nucleotide Degradation - END PRODUCTS

Ammonia + soluble intermediates

Pyrimidine Nucleotide Degradation - PATHWAY OVERVIEW

1. Cytosine → uracil + NH₃ (deamination)

2. Uracil → β-alanine

3. Thymine → β-aminoisobutyrate

4. β-aminoisobutyrate → methylmalonyl semialdehyde → succinyl-CoA

Pyrimidine Nucleotide Degradation - METABOLIC LINK

Succinyl-CoA enters TCA cycle

Pyrimidine Nucleotide Degradation - NITROGEN HANDLING

NH₃ → converted to urea (via urea cycle)

Purine & Pyrimidine Nucleotide Degradation - KEY IDEA

- Purines → uric acid (waste)

- Pyrimidines → metabolic intermediates (reusable

Lesch-Nyhan Syndrome (HGPRT Deficiency) - CAUSE

- Deficiency of HGPRT (Hypoxanthine-Guanine PhosphoribosylTransferase salvage pathway enzyme)

- It recycles purine bases instead of making them from scratch

- Without HGPRT → purines cannot be salvaged

- Purines are NOT recycled; Hypoxanthine & guanine build up → get degraded

Lesch-Nyhan Syndrome (HGPRT Deficiency) - WHAT DOES IT LEAD TO?

Hyperuricemia → severe gout

- B/C Not being used in salvage

- High PRPP stimulates: More purine production → makes problem worse

- Brain relies heavily on: Salvage pathway (NOT de novo)

- Without HGPRT → brain lacks nucleotides → dysfunction

Lesch-Nyhan Syndrome (HGPRT Deficiency) - CLINICAL FEATURES

- Onset ~ 2 years of age

- Poor coordination, neurological dysfunction

- Self-mutilation behavior (biting fingers, lips)

- Cognitive impairment

Gout - CAUSE

- High uric acid/urate levels

- Low solubility → crystal formation

Gout - EFFECTS

- Crystals deposit in joints → inflammation, pain

- Can form kidney/bladder stones

Gout - ASSOCIATED FACTORS

- HGPRT deficiency (↑ purine synthesis)

- Acidic urine or high Ca²⁺

Gout and Its Treatment

Allopurinol

- Converted to alloxanthine

- Inhibits xanthine oxidase

- ↓ Uric acid production

Gout control- KEY CONCEPT

Blocking uric acid formation helps control gout

Severe Combined Immunodeficiency (ADA Deficiency) - CAUSE

Deficiency of adenosine deaminase (ADA)

Severe Combined Immunodeficiency (ADA Deficiency) - BIOCHEMICAL EFFECTS

- Normally ADA helps break down adenosine/deoxyadenosine

- Without ADA → these molecules build up

- The key toxic molecule is: dATP (deoxy-ATP)

- High dATP levels block an important enzyme: ribonucleotide reductase

- No dNTPs → No DNA synthesis → Cells cannot divid

Severe Combined Immunodeficiency (ADA Deficiency) - IMPACT

- Affects rapidly dividing cells → B and T lymphocytes

- Leads to severe immunodeficiency

Severe Combined Immunodeficiency (ADA Deficiency) - KEY CONCEPT

DNA synthesis is essential for immune cell proliferation