Nucleotide Metabolism

Bases/Nucleosides/Nucleotides

Base=

Base Base + Sugar=

Nucleoside

Base + Sugar + Phosphate=

Nucleotide

Adenine DeoxyadenosineDeoxyadenosine 5’-triphosphate

(dATP)

HN

CHN

C

CN

CN

C

NH2

H

NCH

N

NHCN

NH2

O

H

H

HHO

H

H

HOH2C

NCH

N

NHCN

NH2

O

H

H

HHO

H

H

OCH2PO

O

PO

O

P

O- O-O-

O-

O

• Energy metabolism (ATP)*• Monomeric units of nucleic acids*• Regulation of physiological processes

– Adenosine controls coronary blood flow– cAMP and cGMP serve as signaling molecules

• Precursor function-GTP to tetrahydrobiopternin• Coenzyme components- 5’-AMP in FAD/NAD+

• Activated intermediates- UDP Glucose• Allosteric effectors- regulate themselves and others

Cellular Roles of Nucleotides

How I hope to make this at least bearable if not mildly interesting

• Purines and Pyrimidines

– Synthesis (de novo and salvage pathways)

– Degradation

– Relevant disease states

– Relevant clinical applications (Friday)

You are not responsible for any structures

Purines and Pyrimidines

Adenine Guanine

Thymine/Uracil Cytosine

TwoPurines

TwoPyrimidines

HN

CHN

C

CN

CN

C

NH2

H

NC

CC

HN

C

O

CH3

HO

HN

C

CC

N

CH

O

H

H

NH2

HN

CHN

C

CN

CN

C

O

H2N

H

Synthesis Pathways

• For both purines and pyrimidines there are two means of synthesis (often regulate one another)– de novo (from bits and parts)

– salvage (recycle from pre-existing nucleotides)

Salvage Pathwayde novo Pathway

Many Steps Require an Activated Ribose Sugar (PRPP)

5’

de novo Synthesis

• Committed step: This is the point of no return– Occurs early in the biosynthetic pathway– Often regulated by final product (feedback inhibition)

X

Purine Biosynthesis (de novo)

• Atoms derived from:– Aspartic acid– Glycine– Glutamine– CO2

– Tetrahydrofolate

• Also requires– 4 ATP’s

Purines are synthesized on the Ribose ring

Committed Step

Inhibited byAMP, GMP, IMP X

Purine Biosynthesis (de novo)

ATP GTP

(A bunch of steps you don’t need to know)

(Inosine Monophosphate)NCH

NC

CN

CN

C

O

H

NCH

NC

CN

CN

C

O

H2N

H

NCH

NC

CN

CN

C

NH2

HFeedbackInhibition

Purine Degradation

• Sequential removal of bits and pieces

• End product is uric acid• Uric acid is primate-specific

Other species further metabolize

uric acid

Excreted inUrine

XanthineOxidase

Excess Uric Acid Causes Gout

• Primary gout (hyperuricemia)– Inborn errors of metabolism that lead to overproduction of Uric Acid

• Overactive de novo synthesis pathway– Leads to deposits of Uric Acid in the joints– Causes acute arthritic joint inflammation

Offal foods such as liver, kidneys, tripe, sweetbreads and tongueAvoid:

XanthineOxidase

Allopurinol

X

Immunodeficiency Diseases Associated with Purine Degradation

• Defect in adenosine deaminase– Removes amine from adenosine

• SCID- severe combined immunodeficiency

• “Bubble Boy” Disease• Defect in both B-cells and T-

cells (Disease of Lymphocytes)• Patients extremely susceptible to

infection - hence the Bubble

Lymphocyte

Therapies for SCID

• Can be diagnosed in infants through a simple blood test (white cell count)

• Bone marrow transplant for infants – Familial donor

• Continued administration of adenosine deaminase (ADA-PEG)

• Gene therapy- repair defective gene in T-cells or blood stem cells

Salvage Pathway for Purines

Hypoxanthineor

Guanine

+ PRPP = IMP or GMP + PPi Hypoxanthineguanosylphosphoribosyl transferase

(HGPRTase)

Adenine + PRPP = AMP + PPi Adeninephosphoribosyl transferase

(APRTase)

Lesch-Nyhan Syndrome

• Absence of HGPRTase• X-linked (Gene on X)

– Occurs primarily in males

• Characterized by:– Increased uric acid

– Spasticity

– Neurological defects

– Aggressive behavior

– Self-mutilation

Total Aside on X-linked Diseases

• Why are X-linked diseases generally found only in males?

• Females have two X chromosomes - would need to mutate both copies to see a recessive phenotype

• Males have a single X chromosome

XY XX

Think about Fragile X Syndrome

Biosynthesis of Pyrimidines

• Pyrimidine rings are synthesized independent of the ribose and transferred to the PRPP (ribose)

• Generated as UMP (uridine 5’-monophosphate)

• Synthesized from:– Glutamine

– CO2

– Aspartic acid– Requires ATP

NC

CC

HN

C

O

CH3

HO

H

NC

CC

N

CH

O

H

H

NH2

Uracil Cytosine

Regulation of Pyrimidine Biosynthesis

• Regulation occurs at first step in the pathway (committed step)

• 2ATP + CO2 + Glutamine = carbamoyl phosphate

Inhibited by UTPIf you have lots of UTP around this means you won’t

make more that you don’t need

X

Hereditary Orotic Aciduria

• 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

Why does UMP Cure Orotic Aciduria?

CarbamoylPhosphate Orotate

UMPSynthetase

X

FeedbackInhibition

• Disease (-UMP)– No UMP/excess orotate

• Disease (+UMP)– Restore depleted UMP– Downregulate pathway via feedback inhibition (Less orotate)

Biosynthesis: Purine vs Pyrimidine

• Synthesized on PRPP

• Regulated by GTP/ATP• Generates IMP• Requires Energy

• Synthesized then added to PRPP

• Regulated by UTP• Generates UMP/CMP• Requires Energy

Both are very complicated multi-step process whichyour kindly professor does not expect you to know in detail

Pyrimidine Degradation/Salvage

• Pyrimindine rings can be fully degraded to soluble structures (Compare to purines that make uric acid)

• Can also be salvaged by reactions with PRPP– Catalyzed by Pyrimidine phosphoribosyltransferase

Degradation pathways are quite distinct for purines and pyrimidines, but salvage pathways are quite similar

Wait a minute:So far we’ve only made GMP, AMP, and UMP

We need the dNTPs according to the Know-it-All Professor who

taught us that a couple of months ago

Two Problems

• These are monophosphates (i.e. GMP)- we need triphosphates (i.e. GTP) for both DNA and RNA synthesis

• These are ribonucleotides- that’s fine for RNA but we also need to make DNA

Synthesis of ribonucleotides first supports the RNA world theory

Specific Kinases Convert NMP to NDP

NucleosideMonophosphates

NucleosideDiphosphates

MonophosphateKinases

• Monophosphate kinases are specific for the bases

AMP + ATP 2ADP

GMP + ATP GDP + ADP

Adenylate Kinase

Guanylate Kinase

Conversion of Ribonucleotides to Deoxyribonucleotides

OH

HHO

H

H

HOCH2

OH

OH

1´

2´3´

4´5´O

H

HHO

H

H

HOCH2 OH

H

1´

2´3´

4´

5´BASE BASE

Deoxyribonucleoside Ribonucleoside

Somehow we need to get rid of this oxygen

RibonucleotideReductase

Ribonucleotide Reductase

• Catalyzes conversion of NDP to dNDP

• Highly regulated enzyme

• Regulates the level of cellular dNTPs

• Activated prior to DNA synthesis

• Controlled by feedback inhibition

dNDP to dNTP (the final step)

• Once dNDPs are generated by ribonucleotide reductase a general kinase can phosphorylate to make the dNTP’s

• So far we’ve made GTP, ATP, and UTP (which can be aminated to form CTP)

• What about TTP?

You’ll have to tune in tomorrow

Plan for Tomorrow

• Brief Explanation of how dUMP is converted to dTMP

• Some clinically relevant treatments based on these pathways that are used to combat:– Cancer– Bacterial Infections– Viral Infections

Take Home Concepts from Today’s Lecture

• Nucleotides can be made through two pathways – (de novo and salvage)

• Pathways are regulated by feedback inhibition

• Specific degradation pathways exist

• Molecular basis of metabolic diseases mentioned

• What steps are necessary to generate a dNTP from the initial NMP made