NUCLEOTIDE METABOLISM

LECTURE 41 &42

Objectives

At the end of these lectures, students should be able

to:

• Describe synthesis of deoxyribonucleotides

• Compare purine and pyrimidine synthesis

• Compare purine and pyrimidine degradation

Both the salvage and de novo synthesis pathways of purine

and pyrimidine biosynthesis lead to production of

nucleoside-5'-phosphates through the utilization of an

activated sugar intermediate and a class of enzymes called

phosphoribosyltransferases.

The activated sugar used is 5-phosphoribosyl-1-

pyrophosphate, PRPP.

PRPP is generated by the action of PRPP synthetase and

requires energy in the form of ATP as shown:

Nucleotide Metabolism

Nucleotide Metabolism

There are two distinct pathways leading to purines:

1. The de novo pathway (from scratch)

2. Salvage pathways (take free base and create a nucleotide)

3. Conversion of ribonucleotides into deoxyribonucleotides

The major site of purine synthesis is in the liver.

Dietary absorption of ingested purines and pyrimidines is very

low (most of the purines are converted to uric acid in the

intestine)

Purine Synthesis

Origin of the atoms in the purine ring

Synthesis of the purine nucleotides begins with PRPP and

leads to the first fully formed nucleotide, inosine 5'-

monophosphate (IMP)

Purine Synthesis

Synthesis of Inosine Monophosphate

• Basic pathway for biosynthesis of purine ribonucleotides

• Starts from ribose-5-phosphate which is derived from the

pentose phosphate pathway

• Requires 11 steps overall

• occurs primarily in the liver

Steps 1 thru 3

• Step 1:Activation of ribose-5-phosphate – enzyme: PRPP synthetase

– product: 5-phosphoribosyl-a-pyrophosphate (PRPP)

– PRPP is also a precursor in the biosynthesis of pyrimidine

nucleotides and the amino acids histidine and tryptophan

Step 2: commited step

Acquisition of purine atom 9

enzyme: Gln: PRPP amido transferase

displacement of pyrophosphate group by glutamine amide nitrogen

product: b-5-phosphoribosylamine

Steps 1 and 2 are tightly regulated

by feedback inhibition

Steps 3

Acquisition of purine atoms C4,

C5, and N7

– enzyme: glycinamide

ribotide synthetase

– b-phosphoribosylamine

reacts with ATP and glycine

– product: glycinamide

ribotide (GAR)

Steps 4 thru 6

Step 4: acquisition of purine atom C8

– formylation of free a-amino group of GAR

– enzyme: GAR transformylase

– co-factor of enzyme is N10-formyl THF

Step 5: acquisition of purine atom N3

– The amide amino group of a second glutamine is transferred to form formylglycinamidine ribotide (FGAM)

Step 6:

• closing of the imidazole ring or formation of 5-aminoimidazole ribotide

Step 7

Acquisition of C6

– C6 is introduced as HCO3-

– enzyme: AIR carboxylase (aminoimidazole ribotide

carboxylase)

– product: CAIR (carboxyaminoimidazole ribotide)

– enzyme composed of 2 proteins: PurE and PurK (synergistic

proteins)

Steps 8

Acquisition of N1

– N1 is acquired from

aspartate in an amide

condensation reaction

– enzyme: SAICAR

synthetase

– product: 5-

aminoimidazole-4-(N-

succinylocarboxamide)ri

botide (SAICAR)

– reaction is driven by

hydrolysis of ATP

Step 9: Elimination of fumarate

– Enzyme: adenylosuccinate lyase

– Product: 5-aminoimidazole-4-carboxamide ribotide (AICAR)

Step 10: Acquisition of C2

– Another formylation reaction catalyzed by AICAR transformylase

– Product: 5-formaminoimidazole-4-carboxamide ribotide

(FAICAR)

Step 11

Cyclization or ring closure to form IMP

• water is eliminated

• in contrast to step 6 (closure of the imidazole ring), this reaction does not require ATP hydrolysis

• once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells

Outline of Purine

Ring Biosynthesis

10 steps are required:

Two ring closure

Biosynthesis of AMP from IMP

Notes

1- GTP is required for AMP synthesis

2- Need to add –NH2 group to

position six of IMP; use the succinate

to fumarate reaction (urea cycle)

3- Adenylosuccinate synthetase is

regulated by AMP levels

Biosynthesis of GMP from IMP

Conversion of AMP, GMP to more

Phosphorylated Form

Conversion of IMP to AMP and GMP

- to be incorporated into DNA and RNA, nucleoside

monophosphates (NMP’s) must be converted into

nucleoside triphosphates (NTP’s)

- nucleoside monophosphate kinases (adenylate & guanylate kinases)

- nucleoside diphosphate kinase

AMP + ATP 2 ADP

GMP + ATP GDP + ADP

accomplished by separate enzymes

GDP + ATP GTP + ADP

same enzyme acts on all nucleotide di & triphosphates

nucleoside diphosphate kinase is an enzyme which plays

a key role in the activation of antiviral nucleosides such as Retrovir/AZT

Purine nucleoside diphosphates and triphosphates

Regulatory Control of Purine

Nucleotide Biosynthesis • GTP is involved in AMP synthesis and ATP is involved in

GMP synthesis (reciprocal control of production)

• PRPP is a biosynthetically “central” molecule (why?)

– ADP/GDP levels – negative feedback on Ribose Phosphate

Pyrophosphokinase

– Amidophosphoribosyl transferase is activated by PRPP levels

– APRT activity has negative feedback at two sites

• ATP, ADP, AMP bound at one site

• GTP,GDP AND GMP bound at the other site

• Rate of AMP production increases with increasing

concentrations of GTP; rate of GMP production increases with

increasing concentrations of ATP

Regulatory Control of Purine Biosynthesis

• Above the level of IMP production:

– Independent control

– Synergistic control

– Feedforward activation by PRPP

• Below level of IMP production

– Reciprocal control

• Total amounts of purine nucleotides controlled

• Relative amounts of ATP, GTP controlled

The purine salvage pathway

• Purine bases created by degradation of RNA or DNA and intermediate of purine synthesis were costly for the cell to make, so there are pathways to recover these bases in the form of nucleotides

• Two phosphoribosyl transferases are involved:

– APRT (adenine phosphoribosyl transferase) for adenine

– HGPRT (hypoxanthine guanine phosphoribosyl transferase) for guanine or hypoxanthine

Salvage of purines

O

HH

OHOH

CH2

H O

OP

O

OH

OH

H

P

O

O-

O P

O

O-

O-

O

HH

OHOH

CH2

H H

OP

O

OH

OH N N

NN

NH2

adenine

PPi

Adenine

phosphoribosyltransferase

(APRT)

Salvage of purines

• Salvage is needed to maintain the purine pool (biosynthesis is

not completely adequate, especially in neural tissue)

• Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)

• Hypoxanthine + PRPP IMP + Ppi

• Guanine + PRPP GMP + Ppi

• Lack of HGPRT leads to Lesch-Nyhan syndrome. Lack of

enzyme leads to overproduction of purines which are

metabolized to uric acid, which damages cells

Salvage of purine bases

Synthesis of Pyrimidine Ribonucleotides

• Shorter pathway than for purines

• Base is made first, then attached to ribose-P (unlike purine biosynthesis)

• Only 2 precursors (aspartate and glutamine, plus HCO3

-) contribute to the 6-membered ring

• Requires 6 steps (instead of 11 for purine)

• The product is UMP (uridine monophosphate)

Origin of the atoms in the pyrimidine ring

Pyrimidine Biosynthesis

Important differences from Purine Biosynthesis: 1- Pyrimidine base is syntheized before the ribose is added

2- PRPP, Gln, CO2, and aspartate are required for both purine and

pyrimidine biosynthesis

3- There is both de novoand salvage pathways for pyrimidine

synthesis, although the salvage pathway is not as important as it is

for purine recycling.

Step 1: Synthesis of carbamoyl phosphate

Condensation of glutamine, bicarbonate in the presence of ATP

Carbamoyl phosphate synthetase exists in 2 types: CPS-I which

is a mitochondrial enzyme and is dedicated to the urea cycle

and arginine biosynthesis) and CPS-II, a cytosolic enzyme

used here

CPS-II is the major site of regulation in animals: UDP and

UTP inhibit the enzyme and ATP and PRPP activate it

It is the committed step in animals

Step 2: Synthesis of Carbamoyl Aspartate

Enzyme is aspartate transcarbamoylase (ATCase),

catalyzes the condensation of carbamoyl phosphate with aspartate

with the release of Pi

ATCase is the major site of regulation in bacteria; it is activated by

ATP and inhibited by CTP

Carbamoyl phosphate is an “activated” compound, so no energy

input is needed at this step

Step 3: Ring closure to form Dihydroorotate

Enzyme: dihydroorotase

Forms a pyrimidine from

carbamoyl aspartate

Water is released in this

process

The first 3 enzymatic reactions are catalyzed by 3 separate proteins/enzymes in E. coli

In animals, all 3 steps are found in a multifunctional enzyme (210 kD). This allows “channeling” of the substrates and products between active sites without releasing them to the medium where they could be degraded.

The acronym CAD is used as a name for the multienzyme: carbamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroorotase

Channeling also increases the overall rate of multistep processes

Step 1-3: Pyrimidine Synthesis

Step 4: Oxidation of Dihydroorotate to Orotate

An irreversible reaction

Enzyme: dihydroorotate dehydrogenase

Oxidizing power is derived from quinones (thru

coenzyme Q)

Step 5: Acquisition of Ribose phosphate moiety

Enzyme: orotate phosphoribosyl transferase

Ribose phosphate originates from PRPP

Product is orotidine-5’-monophosphate (OMP)

Orotate phosphoribosyl transferase is also used in salvage of uracil and cytosine to their corresponding nucleotide

Step 6: Decarboxylation of OMP

Enzyme: OMP decarboxylase

Product: uridine monophosphate (UMP)

In animals, steps 5 and 6 are catalyzed by a single polypeptide with 2 active sites

Regulatory Control of Pyrimidine Synthesis

Differs between bacteria and animals

Bacteria – regulation at ATCase rxn

Animals – regulation at carbamoyl phosphate synthetase II - UDP and UTP inhibit enzyme; ATP and PRPP activate it

- UMP and CMP competitively inhibit OMP Decarboxylase

Purine synthesis inhibited by ADP and GDP at ribose phosphate pyrophosphokinase step, controlling level of PRPP also regulates pyrimidines

Regulation of pyrimidine nucleotide biosynthesis

Glutamine +

HCO3- +

ATP

Carbamoyl

phosphate

carbamoyl phosph.

synthetase

Orotate

OMPUMPUTP + CTP

orotate

phosphoribosyl

transferase

UTP and CTP are feeback inhibitors of CPS II

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

UMP + ATP UDP + ADP

UDP + ATP UTP + ADP

nucleoside diphosphate kinase

CTP synthase (cytidylate synthetase)

N

N

O

H

O

Ribose 3 phosphate

N

NO

Ribose 3 phosphate

NH2glutamine +

ATPGlutamate +

ADP +Pi

CTPUTP

(in bacteria, ammonia donates the amino group)

Pyrimidine base Salvage

The major enzyme is pyrimidine nucleoside phosphorylase, which takes

the free base, + ribose-1-phosphate, to produce the nucleoside and free

phosphate. This enzyme has a very low affinity for thymine, so never

make a ribo-thyamine.

- Thymine phosphorylase will take thymine + deoxy-

ribose-1-phosphate to produce thymidine

This is different from purine nucleoside phosphorylase, which degraded

the nucleoside to the free base + ribose-1-phosphate

Once the nucleosides are formed, kinases will phosphorylate them

Uridine-Cytidine kinase thakes either uridine or cytidine,

plus ATP, to yield UMP or CMP plus ADP

Thymidine kinase takes thymidine plus ATP to make

dTMP plus ADP

Deoxycytidine kinase takes dC +ATP to produce dCMP

and ADP

This enzyme has low

affinity for thyamine,so

the thyamine

ribonucleotide is rarely, if

ever, produce

Pyrimidine base Salvage

Formation of Deoxyribonucleotides

O

HH

OHOH

CH2

H H

OP

O

OH

OHBase

O

HH

HOH

CH2

H H

OP

O

OH

OHBase

ribonucleotide reductase

dADP, dGDP, dUDP and dCDP are all synthesized by the same enzyme

Synthesized from nucleoside diphosphate (not mono or triphosphate) by

ribonucleotide reductase

All pathways shown previously led to Synthesis of Ribonucleotides

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 which

your kindly professors do not expect you to know in detail

Purine Catabolism • All purine degradation leads to uric acid (but it might not stop

there)

• Ingested nucleic acids are degraded to nucleotides by pancreatic nucleases, and intestinal phosphodiesterases in the intestine

• Group-specific nucleotidases and non-specific phosphatases degrade nucleotides into nucleosides

– Direct absorption of nucleosides

– Further degradation

Nucleoside + H2O base + ribose (nucleosidase)

Nucleoside + Pi base + r-1-phosphate (n. phosphorylase)

NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND EXCRETED.

Intracellular Purine Catabolism

• Nucleotides broken into nucleosides by action

of 5’-nucleotidase (hydrolysis reactions)

• Purine nucleoside phosphorylase (PNP)

– Inosine Hypoxanthine

– Xanthosine Xanthine

– Guanosine Guanine

– Ribose-1-phosphate splits off

• Can be isomerized to ribose-5-phosphate

• Adenosine is deaminated to Inosine (ADA)

Intracellular Purine Catabolism

• Xanthine is the point of convergence for the

metabolism of the purine bases

• Xanthine Uric acid

– Xanthine oxidase catalyzes two reactions

• Purine ribonucleotide degradation pathway is

same for purine deoxyribonucleotides

Adenosine Degradation

Xanthosine Degradation

• Ribose sugar gets recycled (Ribose-1-Phosphate R-5-P )

– can be incorporated into PRPP (efficiency)

• Hypoxanthine is converted to Xanthine by Xanthine Oxidase

• Guanine is converted to Xanthine by Guanine Deaminase

• Xanthine gets converted to Uric Acid by Xanthine Oxidase

AMP + H2O IMP + NH4+ (AMP Deaminase)

IMP + Aspartate + GTP AMP + Fumarate + GDP + Pi

(Adenylosuccinate Synthetase)

COMBINE THE TWO REACTIONS:

Aspartate + H2O + GTP Fumarate + GDP + Pi + NH4+

The overall result of combining reactions is deamination of Aspartate to Fumarate at the expense of a GTP

THE PURINE NUCLEOTIDE CYCLE

- The purine nucleotide cycle for

anaplerotic replenishment of

citric acid cycle intermediates in

skeletal muscle

- Occurs primarily in muscles

and brain (absent in most other

tissues).

- As AMP levels increase during

exercise, the cycle begins

-The ammonia produced can

buffer lactic acid, or can be

incorporated into glutamine

-Fumarate used as intermediate

for TCA

Uric Acid Excretion

• Humans – excreted into urine as insoluble

crystals

• Birds, terrestrial reptiles, some insects –

excrete insoluble crystals in paste form

– Excess amino N converted to uric acid

• (conserves water)

• Others – further modification :

Uric Acid Allantoin Allantoic Acid Urea Ammonia

Gout

• Impaired excretion or overproduction of uric acid

• Uric acid crystals precipitate into joints (Gouty Arthritis), kidneys, ureters (stones)

• Lead impairs uric acid excretion – lead poisoning

• Usually affect joints in the lower extremities (the big toe is the classic site)

• Xanthine oxidase inhibitors inhibit production of uric acid, and treat gout

• Allopurinol treatment – hypoxanthine analog that binds to Xanthine Oxidase to decrease uric acid production

Degradation of Pyrimidines

• CMP and UMP degraded to bases similarly to purines

– Dephosphorylation

– Deamination

– Glycosidic bond cleavage

• Uracil reduced in liver, forming b-alanine

– Converted to malonyl-CoA fatty acid synthesis for energy metabolism

Degradation of Pyrimidines

Note

1- Occurs in liver

2- End products soluble

(unlike purine degradation)

3- No associated disorders