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

Nucleotide Metabolism
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-1pyrophosphate, PRPP. PRPP is generated by the action of PRPP synthetase and requires energy in the form of ATP as shown:

Nucleotide Metabolism

Purine Synthesis
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)

Origin of the atoms in the purine ring

Purine Synthesis
Synthesis of the purine nucleotides begins with PRPP and leads to the first fully formed nucleotide, inosine 5'monophosphate (IMP)

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 5aminoimidazole 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: 5aminoimidazole-4-(Nsuccinylocarboxamide)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

Purine nucleoside diphosphates and triphosphates

- to be incorporated into DNA and RNA, nucleoside monophosphates (NMPs) must be converted into nucleoside triphosphates (NTPs) - nucleoside monophosphate kinases (adenylate & guanylate kinases)
AMP + ATP 2 ADP accomplished by separate enzymes GMP + ATP GDP + ADP

- nucleoside diphosphate kinase

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

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
OH OH P O H OH OH O CH2 H H O H O O P OO O P OOPPi adenine

NH2 N N N

Adenine phosphoribosyltransferase (APRT)

OH OH P O H OH OH O CH2 H O H

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

Step 1-3: Pyrimidine Synthesis

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 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 + carbamoyl phosph. HCO3- + synthetase ATP

Carbamoyl phosphate

Orotate orotate phosphoribosyl transferase

UTP + CTP

UMP

OMP

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 + ATP

UDP + ADP UTP + ADP

nucleoside diphosphate kinase

CTP synthase (cytidylate synthetase) O H N O N Ribose UTP

3

glutamine + ATP

Glutamate + ADP +Pi N O

NH2

N Ribose CTP
3

phosphate

phosphate

(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 + deoxyribose-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

Pyrimidine base Salvage

This enzyme has low affinity for thyamine,so the thyamine ribonucleotide is rarely, if ever, produce

Formation of Deoxyribonucleotides
All pathways shown previously led to Synthesis of Ribonucleotides
ribonucleotide reductase OH OH P O H OH OH O CH2 H O H H Base OH

OH P O H OH H O CH2 H O H

Base

dADP, dGDP, dUDP and dCDP are all synthesized by the same enzyme Synthesized from nucleoside diphosphate (not mono or triphosphate) by ribonucleotide reductase

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

THE PURINE NUCLEOTIDE CYCLE

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 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