Nucleotides: Synthesis and Degradation

Tapeshwar Yadav(Lecturer)BMLT, DNHE, M.Sc. Medical Biochemistry

Nitrogenous BasesPlanar, aromatic, and heterocyclicDerived from purine or pyrimidineNumbering of bases is “unprimed”

Nucleic Acid BasesPurines Pyrimidines

SugarsPentoses (5-C sugars)Numbering of sugars is “primed”

Sugars D-Ribose and 2’-Deoxyribose

*Lacks a 2’-OH group

NucleosidesPurine or pyrimidine base + Sugar

through an N-glycosidic linkagePurines bind to the C1’ carbon of the

sugar at their N9 atomsPyrimidines bind to the C1’ carbon of

the sugar at their N1 atoms.

Nucleosides

Phosphate GroupsMono-, di- or triphosphates

Phosphates bind at C3 or C5 atoms of the sugar

NucleotidesResult from linking one or more phosphates

with a nucleoside onto the 5’ end of the molecule through esterification

NucleotidesRNA (ribonucleic acid) is a polymer of

ribonucleotidesDNA (deoxyribonucleic acid) is a

polymer of deoxyribonucleotidesBoth deoxy- and ribonucleotides contain

Adenine, Guanine and CytosineRibonucleotides contain UracilDeoxyribonucleotides contain Thymine

NucleotidesMonomers for nucleic acid polymersNucleoside Triphosphates are important

energy carriers (ATP, GTP)cAMPImportant components of coenzymes

FAD, NAD+ and Coenzyme A

Naming ConventionsNucleosides:

Purine nucleosides end in “-sine” Adenosine, Guanosine

Pyrimidine nucleosides end in “-dine”Thymidine, Cytidine, Uridine

Nucleotides:Start with the nucleoside name from above

and add “mono-”, “di-”, or “triphosphate”Adenosine Monophosphate, Cytidine

Triphosphate, Deoxythymidine Diphosphate

Digestion of Nucleic acids

Nucleotide MetabolismPURINE RIBONUCLEOTIDES: formed de novo

i.e., purines are not initially synthesized as free bases

First purine derivative formed is Inosine Mono-phosphate (IMP)The purine base is hypoxanthineAMP and GMP are formed from IMP

Most of the tissuesLiverCytosolMulti enzyme complex

Purine Nucleotide Synthesis

OH

H

H

CH2

OH OH

H HO

O2-O3P

-D-Ribose-5-Phosphate (R5P)

O

H

H

CH2

OH OH

H HO

O2-O3P

5-Phosphoribosyl--pyrophosphate (PRPP)

P

O

O

O P

O

O

O

PRPP Synthase

O

H

H

CH2

OH OH

H HO

O2-O3P

5-Phosphoribosyl--pyrophosphate (PRPP)

P

O

O

O P

O

O

O

H

NH2

H

CH2

OH OH

H HO

O2-O3P

-5-Phosphoribosylamine (PRA)

PRPP Glutamyl amido transferase

H

NH2

H

CH2

OH OH

H HO

O2-O3P

-5-Phosphoribosylamine (PRA)

H

NH

H

CH2

OH OH

H HOO2-O3P

CO

H2C NH2

Glycinamide Ribotide (GAR)

GAR Synthetase

H

NH

H

CH2

OH OH

H HOO2-O3P

CO

H2C NH2

Glycinamide Ribotide (GAR)

H2C

CNH

O

CH

HN

O

Ribose-5-Phosphate

Formylglycinamide ribotide (FGAR)

GAR formyl transferaseN5 N10 Methenyl (Formyl) THF

H2C

CNH

O

CH

HN

O

Ribose-5-Phosphate

Formylglycinamide ribotide (FGAR)

H2C

CNH

O

CH

HN

HN

Ribose-5-Phosphate

Formylglycinamidine ribotide (FGAM)

FAGM Synthetase

H2C

CNH

O

CH

HN

HN

Ribose-5-Phosphate

Formylglycinamidine ribotide (FGAM)

HC

CN

CH

N

H2N

Ribose-5-Phosphate

4

5

5-Aminoimidazole Ribotide (AIR)

H2O +AIR Synthetase

HC

CN

CH

N

H2N

Ribose-5-Phosphate

4

5

5-Aminoimidazole Ribotide (AIR)

C

CN

CH

N

H2N

OOC

Ribose-5-Phosphate

4

5

Carboxyamidoimidazole Ribotide (CAIR)

AIR Carboxylase

C

CN

CH

N

H2N

OOC

Ribose-5-Phosphate

4

5

Carboxyamidoimidazole Ribotide (CAIR)

C

CN

CH

N

H2N

CNH

O

HC

COO

CH2

COO

Ribose-5-Phosphate

4

5

5-Aminoimidazole-4-(N-succinylocarboxamide)ribotide (SAICAR)

SAICAR Synthetase

C

CN

CH

N

H2N

CNH

O

HC

COO

CH2

COO

Ribose-5-Phosphate

4

5

5-Aminoimidazole-4-(N-succinylocarboxamide)ribotide (SAICAR)

Adeno succinate lyase

C

CN

CH

N

H2N

Ribose-5-Phosphate

4

5

5-Aminoimidazole-4-carboxamideribotide (AICAR)

CH2N

O

C

CN

CH

N

NH

Ribose-5-Phosphate

4

5

5-Formaminoimidazole-4-carboxamideribotide (FAICAR)

CH2N

O

CH

O

C

CN

CH

N

H2N

Ribose-5-Phosphate

4

5

5-Aminoimidazole-4-carboxamideribotide (AICAR)

CH2N

O

AICAR Formyl transferase

C

CN

CH

N

NH

Ribose-5-Phosphate

4

5

5-Formaminoimidazole-4-carboxamideribotide (FAICAR)

CH2N

O

CH

O

IMP cyclohydrolase

Inosine Monophosphate (IMP)

HN

HCN

C

CC

N

CH

N

O

4

5

HH

CH2

OH OH

H HOO2-O3P

Purine Nucleotide Synthesis at a Glance

ATP is involved in 6 stepsPRPP in the first step of Purine synthesis is

also a precursor for Pyrimidine Synthesis, His and Trp synthesisRole of ATP in first step is unique– group

transfer rather than couplingIn second step, C1 notation changes from to

(anomers specifying OH positioning on C1 with respect to C4 group)

In step 2, PPi is hydrolyzed to 2Pi (irreversible, “committing” step)

Coupling of ReactionsHydrolyzing a phosphate from ATP is relatively

easy G°’= -30.5 kJ/mol

If endergonic reaction released energy into cell as heat energy, wouldn’t be useful

Must be coupled to an exergonic reactionWhen ATP is a reactant:

Part of the ATP can be transferred to an acceptor: Pi, PPi, adenyl or adenosinyl group

ATP hydrolysis can drive an otherwise unfavorable reaction(synthetase; “energase”)

IMP Conversion to AMP

IMP Conversion to GMP

Regulation of Purine Nucleotide BiosynthesisGTP 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 Pyrophospho synthetase PRPP Glutamyl amido transferase is activated by PRPP

levels

Regulation of Purine Nucleotide BiosynthesisAPRT activity has negative feedback at two sites

ATP, ADP, AMP bound at one siteGTP,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

Regulation of Purine BiosynthesisAbove the level of IMP production:

Independent controlSynergistic controlFeed forward activation by PRPP

Below level of IMP productionReciprocal control

Total amounts of purine nucleotides controlled

Relative amounts of ATP, GTP controlled

PRPP synthetase Activated by PiInhibited by Purine nucleo tides AMP,

GMP and IMP.

Regulation of Purine Biosynthesis

Committed step :PRPP Gluamy amido transferaseActivators

PRPPGlutamine

Inhibitors (Allosteric)AMP, GMP, (IMP)

Synthetic inhibitors :MercaptopurineMetho trexateAzaserineThioguanineAzaguanineSulphonamidesTrimethoprim

Intracellular Purine CatabolismNucleotides broken into nucleosides by

action of 5’-nucleotidase (hydrolysis reactions)

Purine nucleoside phosphorylase (PNP)Inosine HypoxanthineXanthosine XanthineGuanosine GuanineRibose-1-phosphate splits off

Can be isomerized to ribose-5-phosphate

Adenosine is deaminated to Inosine (ADA)

Intracellular Purine CatabolismXanthine is the point of convergence for

the metabolism of the purine basesXanthine Uric acid

Xanthine oxidase catalyzes two reactions

Purine ribonucleotide degradation pathway is same for purine deoxyribonucleotides.

Salvage pathwayRecycling of purines (nucleosides)PRPPTwo enzymesRBCs and Brain.Saves energy expenditure

Purine SalvageAdenine phosphoribosyl transferase (APRT)

Adenine + PRPP AMP + PPi

Hypoxanthine-Guanine phosphoribosyl transferase (HGPRT)

Hypoxanthine + PRPP IMP + PPiGuanine + PRPP GMP + PPi

(NOTE: THESE ARE ALL REVERSIBLE REACTIONS)

AMP,IMP,GMP do not need to be re-synthesized de novo !

Adenosine Degradation

Guanosine 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

Guanosine

Guanine

Xanthine Oxidase A homodimeric proteinContains electron transfer proteins

FADMo-pterin complex in +4 or +6 state Two 2Fe-2S clusters

Transfers electrons to O2 H2O2 H2O2 is toxic Disproportionated to H2O and O2 by

catalase

A CASE STUDY : GOUTA 45 YEAR OLD MAN AWOKE FROM SLEEP WITH A

PAINFUL AND SWOLLEN RIGHT GREAT TOE. ON THE PREVIOUS NIGHT HE HAD EATEN A MEAL OF FRIED LIVER AND ONIONS, AFTER WHICH HE MET WITH HIS POKER GROUP AND DRANK A NUMBER OF BEERS.

HE SAW HIS DOCTOR THAT MORNING, “GOUTY ARTHRITIS” WAS DIAGNOSED, AND SOME TESTS WERE ORDERED. HIS SERUM URIC ACID LEVEL WAS ELEVATED AT 8.0 mg/dL (NL < 7.0 mg/dL).

THE MAN RECALLED THAT HIS FATHER AND HIS GRANDFATHER, BOTH OF WHOM WERE ALCOHOLICS, OFTEN COMPLAINED OF JOINT PAIN AND SWELLING IN THEIR FEET.

A CASE STUDY : GOUTTHE DOCTOR RECOMMENDED THAT THE MAN

USE NSAIDS FOR PAIN AND SWELLING, INCREASE HIS FLUID INTAKE (BUT NOT WITH ALCOHOL) AND REST AND ELEVATE HIS FOOT. HE ALSO PRESCRIBED ALLOPURINOL.

A FEW DAYS LATER THE CONDITION HAD RESOLVED AND ALLOPURINOL HAD BEEN STOPPED. A REPEAT URIC ACID LEVEL WAS OBTAINED (7.1 mg/dL). THE DOCTOR GAVE THE MAN SOME ADVICE REGARDING LIFE STYLE CHANGES.

GoutImpaired excretion or

overproduction of uric acidUric acid crystals precipitate into

joints (Gouty Arthritis), kidneys, ureters (stones)

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

ALLOPURINOL is a XANTHINE OXIDASE inhibitorA substrate ANALOG is converted to an inhibitor.In this case a “SUICIDE-INHIBITOR”

ALCOHOL CONSUMPTION AND GOUT

Causes :1.PRPP Amido transferase2.PRPP Synthetase3.Deficiency of enzymes of salvage

pathway4.Glucose-6-Phosphatase deficiencySecondary hyper urecemia (Gout) :1.Leukemia2.Lymphoma3.Polycythemia4.Trauma5.Starvation6.Renal failure7.Toxemias

Clinical features :ArthritisTophiUrolithiasisRenal failureTreatment :Reduce Purine intake and alcoholProbenecidAllopurinolColchicine

Lesch-Nyhan SyndromeA defect in production or activity of HGPRT.It is an X-linked disorder.

Causes increased level of Hypoxanthine and Guanine ( in degradation to uric acid)

Also PRPP accumulates stimulates production of Purine

nucleotides (and thereby increases their degradation)

Causes gout-like symptoms, but also neurological symptoms spasticity, aggressiveness, self-mutilation

First neuropsychiatric abnormality that was attributed to a single enzyme

Pyrimidine Ribonucleotide Synthesis

Uridine Monophosphate (UMP) is synthesized firstCTP is synthesized from UMP

Pyrimidine ring synthesis completed first; then attached to ribose-5-phosphate

2 ATP + HCO3- + Glutamine + H2O

CO

O PO3-2

NH2

Carbamoyl Phosphate

2 ADP +Glutamate + Pi

CarbamoylPhosphateSynthetase II

Pyrimidine Synthesis

CO

O PO3-2

NH2

Carbamoyl Phosphate

NH2

CNH

CH

CH2

C

COOO

HO

O

Carbamoyl Aspartate

AspartateTranscarbamoylase(ATCase)

Aspartate

Pi

NH2

CNH

CH

CH2

C

COOO

HO

O

Carbamoyl Aspartate

Pyrimidine Synthesis

HN

CNH

CH

CH2

C

COOO

O

Dihydroorotate

HN

CNH

CH

CH2

C

COOO

O

Dihydroorotate

HN

CNH

C

CHC

COOO

O

Orotate

HN

CNH

C

CHC

COOO

O

Orotate

HN

CN

C

CHC

COOO

O

HH

CH2

OH OH

H HOO2-O3P

Orotidine-5'-monophosphate(OMP)

HN

CN

C

CHC

COOO

O

HH

CH2

OH OH

H HOO2-O3P

Orotidine-5'-monophosphate(OMP)

HN

CN

CH

CHC

O

O

HH

CH2

OH OH

H HOO2-O3P

Uridine Monophosphate(UMP)

CO2

OMP Decarboxylase

2 ATP + HCO3- + Glutamine + H2O

CO

O PO3-2

NH2

Carbamoyl Phosphate

NH2

CNH

CH

CH2

C

COOO

HO

O

Carbamoyl Aspartate

HN

CNH

CH

CH2

C

COOO

O

Dihydroorotate

HN

CNH

C

CHC

COOO

O

Orotate

HN

CN

C

CHC

COOO

O

HH

CH2

OH OH

H HOO2-O3P

Orotidine-5'-monophosphate(OMP)

HN

CN

CH

CHC

O

O

HH

CH2

OH OH

H HOO2-O3P

Uridine Monophosphate(UMP)

2 ADP +Glutamate + Pi

CarbamoylPhosphateSynthetase II

AspartateTranscarbamoylase(ATCase)

Aspartate

Pi

H2O

Dihydroorotase

Quinone

ReducedQuinone

DihydroorotateDehydrogenase

PRPP PPi

Orotate PhosphoribosylTransferase

CO2

OMP Decarboxylase

Pyrimidine Synthesis

UMP Synthesis Overview2 ATPs needed: both used in first step

One transfers phosphate, the other is hydrolyzed to ADP and Pi

2 condensation rxns: form carbamoyl aspartate and dihydroorotate (intramolecular)

Dihydroorotate dehydrogenase is an intra-mitochondrial enzyme; oxidizing power comes from quinone reduction

Attachment of base to ribose ring is catalyzed by OPRT; PRPP provides ribose-5-PPPi splits off PRPP – irreversible

Channeling: enzymes 1, 2, and 3 on same chain; 5 and 6 on same chain

OMP DECARBOXYLASE : THE MOST CATALYTICALLY PROFICIENT ENZYME

FINAL REACTION OF PYRIMIDINE PATHWAYANOTHER MECHANISM FOR

DECARBOXYLATIONA HIGH ENERGY CARBANION INTERMEDIATE

NOT NEEDEDNO COFACTORS NEEDED !SOME OF THE BINDING ENERGY BETWEEN

OMP AND THE ACTIVE SITE IS USED TO STABILIZE THE TRANSITION STATE“PREFERENTIAL TRANSITION STATE

BINDING”

UMP UTP and CTPNucleoside 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

Regulation of Pyrimidine Synthesis

Differs between bacteria and animalsBacteria – regulation at ATCase rxn

Animals – regulation at carbamoyl phosphate synthetase IIUDP and UTP inhibit enzyme; ATP and PRPP activate itUMP 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

CPS, ATC & DHOase multi enzyme complexOPRTase & OMP decarboxylase single

functional complex.

Salvage : PRPP and phospho ribosyl transferase

Nucleoside phosphorylase.

Degradation of PyrimidinesCMP and UMP degraded to bases similarly

to purines DephosphorylationDeaminationGlycosidic bond cleavage

Uracil reduced in liver, forming -alanine Converted to malonyl-CoA fatty acid

synthesis for energy metabolism

Deoxyribonucleotide FormationPurine/Pyrimidine degradation are the

same for ribonucleotides and deoxyribonucleotides

Biosynthetic pathways are only for ribonucleotide production

Deoxyribonucleotides are synthesized from corresponding ribonucleotides

Formation of DeoxyribonucleotidesReduction of 2’ carbon done via a free

radical mechanism catalyzed by “Ribonucleotide Reductases”

E. coli RNR reduces ribonucleoside diphosphates (NDPs) to deoxyribonucleoside diphosphates (dNDPs)Two subunits: R1 and R2

A Heterotetramer: (R1)2 and (R2)2 in vitro

ThioredoxinPhysiologic reducing agent of RNRCys pair can swap H atoms with disulfide

formed regenerate original enzymeThioredoxin gets oxidized to disulfide

Oxidized Thioredoxin gets reduced by NADPH ( final electron acceptor)mediated by thioredoxin reductase

Thymine FormationFormed by methylating deoxyuridine

monophosphate (dUMP) UTP is needed for RNA production, but

dUTP not needed for DNAIf dUTP produced excessively, would cause

substitution errors (dUTP for dTTP)dUTP hydrolyzed by dUTPase (dUTP diphosphohydrolase) to dUMP

methylated at C5 to form dTMP rephosphorylate to form dTTP

dUMP dTMP

NADPH + H+

NADP+

SERINE

GLYCINE

REGENERATION OF N5,N10 METHYLENETETRAHYDROFOLATE

DHFN5,N10 – METHYLENE-THF

THF

dihydrofolate reductaseserine hydroxymethyl transferase

thymidylate synthase

dUMP dTMP

NADPH + H+

NADP+

SERINE

GLYCINE

INHIBITORS OF N5,N10 METHYLENETETRAHYDROFOLATE REGENERATION

DHFN5,N10 – METHYLENE-THF

THF

dihydrofolate reductaseserine hydroxymethyl transferase

thymidylate synthase

METHOTREXATE AMINOPTERIN TRIMETHOPRIM

FdUMP

X

X

Anti-Folate DrugsCancer cells consume dTMP quickly for

DNA replicationInterfere with thymidylate synthase rxn to

decrease dTMP production (fluorodeoxyuridylate – irreversible inhibitor) – also

affects rapidly growing normal cells (hair follicles, bone marrow, immune system, intestinal mucosa)

Dihydrofolate reductase step can be stopped competitively (DHF analogs)Anti-Folates: Aminopterin, methotrexate,

trimethoprim

ADENOSINE DEAMINASE DEFICIENCYIN PURINE DEGRADATION, ADENOSINE

INOSINEENZYME IS ADA

ADA DEFICIENCY RESULTS IN SCID“SEVERE COMBINED IMMUNODEFICIENCY”

SELECTIVELY KILLS LYMPHOCYTESBOTH B- AND T-CELLSMEDIATE MUCH OF IMMUNE RESPONSE

ALL KNOWN ADA MUTANTS STRUCTURALLY PERTURB ACTIVE SITE

THE PURINE NUCLEOTIDE CYCLEAMP + 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

Orotic AciduriaCaused by defect in protein chain with

enzyme activities of last two steps of pyrimidine synthesis

Increased excretion of orotic acid in urine

Symptoms: retarded growth; severe anemia

Only known inherited defect in this pathway (all others would be lethal to fetus)

Treat with uridine/cytidine