Nucleic Acids Metabolism

Nitrogenous Bases

• Planar, aromatic, and heterocyclic• Derived from purine or pyrimidine• Numbering of bases is “unprimed”

Nucleic Acid BasesPurines Pyrimidines

Sugars

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

Sugars

D-Ribose and 2’-Deoxyribose

*Lacks a 2’-OH group

Nucleosides

• Result from linking one of the sugars with a purine or pyrimidine base through an N-glycosidic linkage

– Purines bond to the C1’ carbon of the sugar at their N9 atoms

– Pyrimidines bond to the C1’ carbon of the sugar at their N1 atoms

Nucleosides

Phosphate Groups

• Mono-, di- or triphosphates

• Phosphates can be bonded to either C3 or C5 atoms of the sugar

Nucleotides

• Result from linking one or more phosphates with a nucleoside onto the 5’ end of the molecule through esterification

Nucleotides

• RNA (ribonucleic acid) is a polymer of ribonucleotides

• DNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotides

• Both deoxy- and ribonucleotides contain Adenine, Guanine and Cytosine– Ribonucleotides contain Uracil– Deoxyribonucleotides contain Thymine

Nucleotides

• Monomers for nucleic acid polymers• Nucleoside Triphosphates are important

energy carriers (ATP, GTP)• Important components of coenzymes– FAD, NAD+ and Coenzyme A

Naming Conventions

• Nucleosides:– 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

Nucleotide Metabolism• PURINE 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 hypoxanthine• AMP and GMP are formed from IMP

Purine Nucleotides

• Get broken down into Uric Acid (a purine) Buchanan (mid 1900s) showed where purine ring components came from:

N1: Aspartate AmineC2, C8: FormateN3, N9: GlutamineC4, C5, N7: GlycineC6: Bicarbonate Ion

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

ATP

AMP

RibosePhosphatePyrophosphokinase

H

NH2

H

CH2

OH OH

H HO

O2-O3P

-5-Phosphoribosylamine (PRA)

AmidophosphoribosylTransferase

Glutamine + H2O

Glutamate + PPi

H

NH

H

CH2

OH OH

H HO

O2-O3P

CO

H2C NH2

Glycinamide Ribotide (GAR)

GAR Synthetase

Glycine + ATP

ADP+ Pi

H2C

CNH

O

CH

HN

O

Ribose-5-Phosphate

Formylglycinamide ribotide (FGAR)

H2C

CNH

O

CH

HN

HN

Ribose-5-Phosphate

Formylglycinamidine ribotide (FGAM)

THFN10-Formyl-THF

GAR Transformylase

ATP +Glutamine +H2O

ADP +Glutamate + Pi

FGAM Synthetase

HC

CN

CH

N

H2N

Ribose-5-Phosphate

4

5

5-Aminoimidazole Ribotide (AIR)

ATP

ADP + Pi

AIR Synthetase

C

CN

CH

N

H2N

OOC

Ribose-5-Phosphate

4

5

Carboxyamidoimidazole Ribotide (CAIR)

ATP+HCO3

ADP + PiAIR Car boxylase

Aspartate+ ATP

ADP+ Pi

SAICAR Synthetase

AdenylosuccinateLyase

Fumarate

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

C

CN

CH

N

H2N

CNH

O

HC

COO

CH2

COO

Ribose-5-Phosphate

4

5

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

THF

AICAR Transformylase

N10-Formyl-

THF

Inosine Monophosphate (IMP)

HN

HCN

C

CC

N

CH

N

O

4

5

HH

CH2

OH OH

H HOO2-O3P

IMPCyclohydrolase

H2O

Purine Nucleotide Synthesis at a Glance

• ATP is involved in 6 steps

• PRPP in the first step of Purine synthesis is also a precursor for Pyrimidine Synthesis, His and Trp synthesis

– Role of ATP in first step is unique– group transfer rather than coupling

• In second step, C1 notation changes from a to b (anomers specifying OH positioning on C1 with respect to C4 group)

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

• Hydrolyzing a phosphate from ATP is relatively easy G°’= -30.5 kJ/mol

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

– Must be coupled to an endergonic reaction• When 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)

Coupling of Reactions

Purine Biosynthetic Pathway• Channeling of some reactions on pathway organizes and controls

processing of substrates to products in each step– Increases overall rate of pathway and protects intermediates from

degradation• In animals, IMP synthesis pathway shows channeling at:

– Reactions 3, 4, 6– Reactions 7, 8– Reactions 10, 11

IMP Conversion to AMP

IMP Conversion to GMP

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

Purine Catabolism and Salvage

• All purine degradation leads to uric acid • 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

Xanthine Oxidase

• A homodimeric protein• Contains electron transfer proteins– FAD– Mo-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

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

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

Purine Salvage

• Adenine phosphoribosyl transferase (APRT)Adenine + PRPP AMP + PPi

• Hypoxanthine-Guanine phosphoribosyl transferase (HGPRT)

Hypoxanthine + PRPP IMP + PPi

Guanine + PRPP GMP + PPi

(NOTE: THESE ARE ALL REVERSIBLE REACTIONS)

AMP,IMP,GMP do not need to be resynthesized de novo !

Pyrimidine Ribonucleotide Synthesis

• Uridine Monophosphate (UMP) is synthesized first– CTP is synthesized from UMP

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

N1, C4, C5, C6 : AspartateC2 : HCO3

-

N3 : Glutamine amide Nitrogen

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 HO

O2-O3P

Orotidine-5'-monophosphate(OMP)

HN

CN

CH

CHC

O

O

HH

CH2

OH OH

H HO

O2-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 Overview• 2 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-P– PPi splits off PRPP – irreversible

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

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

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

Deoxyribonucleotide Formation

• Purine/Pyrimidine degradation are the same for ribonucleotides and deoxyribonucleotides

• Biosynthetic pathways are only for ribonucleotide production

• Deoxyribonucleotides are synthesized from corresponding ribonucleotides

DNA vs. RNA: REVIEW

• DNA composed of deoxyribonucleotides

• Ribose sugar in DNA lacks hydroxyl group at 2’ Carbon

• Uracil doesn’t (normally) appear in DNA– Thymine (5-methyluracil) appears instead

Formation of Deoxyribonucleotides

• Reduction 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

Thymine Formation

• Formed by methylating deoxyuridine monophosphate (dUMP)

• UTP is needed for RNA production, but dUTP not needed for DNA– If 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