bpt 114. purine and pyrimidine metabolism

Purine and Pyrimidine metabolism

BPT 114: DIGESTIVE, REPRODUCTIVE,ENDOCRINE AND URINARY SYSTEM

LECTURER;Dr. Geoffrey K. MaiyohDepartment of Medical Biochemistry

GKM/MUSOM/BPT 114:DIG.END.REP.URISYS.2014

• Purines and pyrimidines are Nitrogen containing heterocyclic compounds whose rings contain Carbon and Nitrogen.

• The planar character of purines and pyrimidines facilitates their close association or stacking which stabilizes double stranded DNA.

• They are also refereed to as Nitrogenous Bases and are a major components of nucleotides that build DNA and RNA

PURINES AND PYRIMIDINES


Biological functions of nucleotides Building blocks of nucleic acids (DNA and RNA). Involved in energy storage, muscle contraction,

active transport, maintenance of ion gradients. Activated intermediates in biosynthesis

(e.g. UDP-glucose, S-adenosylmethionine). Components of coenzymes (NAD+, NADP+, FAD,

FMN, and CoA) Metabolic regulators:

a. Second messengers (cAMP, cGMP)b. Phosphate donors in signal transduction (ATP)

c. Regulation of some enzymes via adenylation and uridylylation


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


Nucleic Acid Bases

Purines 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 ThymineGKM/MUSOM/BPT

114:DIG.END.REP.URISYS.2014

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 DiphosphateGKM/MUSOM/BPT


Nucleotides

-OO

H(OH)

HH

HHO

OP

O

O-

Purine orPyrimidineBase

Phosphate

Pentose sugar

Nucleoside

Nucleotide

1'

2'3'

4'

5'β-glycosidic bond

RNA- ribose (R)DNA – deoxyribose (dR)


NN

NNH

NH2

NNH

NH

NH

O

O

N

NH

NH2

O

H3C

NH

NH

O

O

XanthineAdenine (A)

Thymine (T)Cytosine (C)

NH

NH

O

O

Uracil (U)

NN

NNH

Purine

N

NH

Pyrimidine

1

2

3

4

5

6

3

2

16

4

57

8

9

NNH

NNH

O

NH2

Guanine (G)

NNH

NNH

O

Hypoxanthine

Nucleobase structures


Hypoxanthine Inosine Inosinate (IMP)Xanthine Xanthosine Xanthylate (XMP)


Two major routes for nucleotide biosynthesis

dNTPs

dNTPs


Synthesis Pathways• For both purines and pyrimidines there are two means

of synthesis (often regulate one another)– de novo (from basic metabolites)

– salvage (recycle from pre-existing nucleotides)

Salvage Pathwayde novo PathwayGKM/MUSOM/BPT 114:DIG.END.REP.URISYS.2014

Purine nucleotides– 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

αO2-O3P

α-D-Ribose-5-Phosphate (R5P)

O

H

H

CH2

OH OH

H HO α

O2-O3P

5-Phosphoribosyl-α-pyrophosphate (PRPP)

P

O

O

O P

O

O

O

ATP

AMP

RibosePhosphatePyrophosphokinase

H

NH2

H

CH2

OH OH

H HO

βO2-O3P

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

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


de novo Synthesis and regulation• Committed step: This is the point of no return

– Occurs early in the biosynthetic pathway– Often regulated by final product (feedback

inhibition)

X


• 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: P i, PPi, adenyl, or adenosinyl group

– ATP hydrolysis can drive an otherwise unfavorable reaction

(synthetase; “energase”)

Coupling of Reactions


Substrate Channeling• Substrate channeling is the process of direct

transfer of an intermediate between the active sites of two enzymes that catalyze sequential reactions in a biosynthetic pathway.

• The active sites can be located either on separate domains in a multifunctional enzyme or on separate subunits in a multienzyme complex.


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, 11GKM/MUSOM/BPT


IMP Conversion to AMP and GMP

Regulatory Control of Purine Nucleotide Biosynthesis

• GTP is involved in AMP synthesis and ATP is involved in GMP synthesis (reciprocal control of production)

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Regulatory Control of Purine Biosynthesis

• Above the level of IMP production:– Independent control– Synergistic control

– Feed forward activation by PRPP

• Below level of IMP production– Reciprocal control

Overall• Total amounts of purine nucleotides is controlled• Relative amounts of ATP, GTP controlled


Purine Catabolism and Salvage• 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.


GMO phobia


We can not even absorb whole nucleotides, how then do we absorb a gene???

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


Major pathways of purine catabolism in animals.

ADA

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 !


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 from pewter drinking goblets– Fall of Roman Empire?

• 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 INHIBITOR

A SUBSTRATE ANALOG IS CONVERTED TO AN INHIBITOR, IN THIS CASE A “SUICIDE-INHIBITOR”


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

β

Orotidine-5'-monophosphate(OMP)

HN

CN

CH

CHC

O

O

HH

CH2

OH OH

H HO

O2-O3P

β

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


53

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

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


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


Orotic Aciduria• Caused 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 • IN-CLASS QUESTION: HOW DOES URIDINE

AND CYTIDINE ADMINISTRATION WORK TO TREAT OROTIC ACIDURIA?


How does UMP/UTP Cure Orotic Aciduria?

UMPSynthetase

XCarbamoylPhosphate Orotate

FeedbackInhibition

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

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

55

Degradation of Pyrimidines

• CMP and UMP degraded to bases similarly to purines – Dephosphorylation– Deamination– Glycosidic bond cleavage

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

synthesis for energy metabolism


Thank you for your attention


bpt 114. purine and pyrimidine metabolism

Health & Medicine