METABOLISME NUKLEOTIDA

PURIN dan PIRIMIDIN

Dr. Marhaen Hardjo, M.Biomed, PhD

Bagian Biokimia Fakultas Kedokteran

Universitas Hasanuddin Makassar 1
 METABOLISME
NUKLEOTIDA PURIN dan PIRIMIDIN :
* KONSUMSI NUKLEOTIDA KEBANYAKAN BERUPA
NUKLEOPROTEIN.
* MANUSIA MAMPU MENSINTESIS NUKLEOTIDA SECA
RA DE NOVO DARI SENYAWA AMFIBOLIK.
PENCERNAAN NUKLEOTIDA PURIN dan PIRIMIDIN :
NUKLEOTIDA PURIN dan PIRIMIDIN DIPECAH
OLEH ENZIM-ENZIM DALAM Tr. DIGESTIVUS.
HASIL AKHIRNYA :
-. ASAM URAT
-. CO2, NH3 dan ASAM - ISOBUTIRAT
 NUKLEOPROTEIN dari MAKANAN
ENZIM PROTYEOLITIK &
DNAase & RNAase
POLINUKLEOTIDA = ASAM NUKLEAT + PROTEIN

POLINUKLEOTIDASE

( MONO ) NUKLEOTIDA

NUKLEOTIDASE & FOSFATASE

NUKLEOSIDA + FOSFAT
NUKLEOSIDASE

PURIN dan PIRIMIDIN + PENTOSA

( RIBOSA / DEOKSIRIBOSA )

ASAM URAT CO2 + NH3 + ASAM--ISOBUTIRAT

Hampir semua organisme mampu
mensintesis nukleotida dr prekursor yg
lebih sederhana jalur de novo untuk
nukleotida mirip utk setiap organisme

Nukleotida juga dapat disintesis dari hasil

pemecahan nukleotida yang telah ada
salvage pathway (recycle) yaitu dari
degradasi pirimidin dan purin dari sel
yang mati (regenerasi) atau dari makanan
 Degradasi nukleotida

Di dalam usus halus tjd pemutusan ikatan fosfodiester

oleh endonuklease (pankreas) oligonukleotida
Dipecah lebih lanjut dg fosfodiesterase (ensim
exonuclease non spesifik) monofosfat
Dipecah lbh lanjut fosfomonoesterase dikenal sebagai
nukleotidase menghasilkan nukleosida and
orthophosphate.
Nucleosida phosphorylase menghasilkan basa dan
and ribose-1-phosphate.
Jika basa atau nukleosida tidak digunakan kembali utk
salvage pathways, basa akan lebih lanjut didegradasi

asam urat ureidopropionat

(purin) (pyrimidine).
 Purine Nucleotides
Get broken down into Uric Acid (a purine)
Buchanan (mid 1900s) showed where purine
ring components came from:

N1: Aspartate Amine

C2, C8: Formate
N3, N9: Glutamine
C4, C5, N7: Glycine
C6: Bicarbonate Ion
KATABOLISME PURIN :
HASIL AKHIR KATABOLISME PURIN ADALAH ASAM URAT.
99 % ASAM URAT MERUPAKAN HASIL PEMECAHAN OLEH
ENZIM NUKLEOSIDA PURIN FOSFORILASE .
REAKSINYA :
ADENOSIN GUANOSIN

ADENOSIN PURIN
DEAMINASE -NUKLEOSIDA
-FOSFORILASE
INOSIN GUANIN

GUANASE
PURIN NUKLEOSIDA
OKSIDASE
HIPOXANTIN XANTIN
XANTIN OKSIDASE
XANTIN
-OKSIDASE

ASAM URAT
( SUKAR LARUT )
 Degradasi purine

Produk akhir katabolisme purin : asam urat

Vertebrata terestrial
urea ureotelic
Burung & reptil
asam urat
uricotelic
Binatang di air
ammonia
ammonotelic
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.
 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
 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
 Purine Nucleotide Cycle
***
In-Class Question: Why is the purine nucleotide
cycle important in muscle metabolism during a
burst of activity?
 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

KATABOLISME PIRIMIDIN :
HASIL AKHIRNYA adalah CO2, NH3 dan ASAM - AMINO
BUTIRAT, YANG MUDAH LARUT.
SITOSIN TIMIN

NH3

URASIL DIHIDROTIMIN
NADPH + H+
NADP+
DIHIDROURASIL -URIDOISOBUTIRAT

H2 O
ASAM UREIDOPROPIONAT
H2 O
ALANIN
- AMINO BUTIRAT

CO2 + NH3
 Degradation of Pyrimidines
CMP and UMP degraded to bases
similarly to purines
Dephosphorylation
Deamination
Glycosidic bond cleavage
Uracil reduced in liver, forming -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
 Degradasi Pirimidin

1.Membentuk produk larut dalam air CO, NH, -Alanin dan -

Aminoisobutirat
2.Ekresi -Aminoisobutirat meningkat pada Leukemia dan
pajanan radiasi sinar-X yang mengakibatkan perusakan DNA
Degradasi
pirimidin
Sintesis Purin
 Purine Nucleotide Synthesis COO
O

OOC C
2-
O3P O CH2 H N HC N N
O C4 Aspartate ADP C4
H H CH + ATP + Pi
H
CH
5 CH2 5
H C C
OH N N
OH OH H2N SAICAR Synthetase COO H2N
-D-Ribose-5-Phosphate (R5P)
Ribose-5-Phosphate Ribose-5-Phosphate
ATP Carboxyamidoimidazole Ribotide (CAIR) 5-Aminoimidazole-4-(N-succinylocarboxamide)
Ribose
Phosphate ribotide (SAICAR)
ADP + Pi
Pyrophosphokinase AIR
Car boxylase
AMP Fumarate Adenylosuccinate
ATP Lyase
+HCO3 O
N
2-
O3P O CH2 H HC 4 C
O O H2N N
O CH
H H C4
5
H O P O P O C CH
OH N C
5
OH
O O
H2N N
H2N
Ribose-5-Phosphate
Ribose-5-Phosphate
5-Aminoimidazole Ribotide (AIR)
5-Phosphoribosyl--pyrophosphate (PRPP) 5-Aminoimidazole-4-carboxamide
ADP + Pi ribotide (AICAR)
AIR
Glutamine Synthetase N10-Formyl-
+ H2O Amidophosphoribosyl THF
ATP AICAR
Transferase
Transformylase
Glutamate H O THF
+ PPi N
H2C CH C
H2N N
2- C4
O3P O CH2 NH2
O CH
H H C O 5
HN NH C
N
H H O C NH
OH OH H
Ribose-5-Phosphate Ribose-5-Phosphate
-5-Phosphoribosylamine (PRA)
Formylglycinamidine ribotide (FGAM) 5-Formaminoimidazole-4-carboxamide
Glycine ADP + ribotide (FAICAR)
+ ATP Glutamate + Pi
FGAM
GAR Synthetase H2O
Synthetase IMP
ATP + Cyclohydrolase
ADP
Glutamine +
+ Pi H2O O
H
H2C NH2 N C N
H2 C CH HN C
4
O C CH
HC C5
2-
C O N
O3P O CH2 NH N
O N10-Formyl-THF THF O NH 2-
O3P O CH2
H H O
H H
H H H
OH
GAR Transformylase Ribose-5-Phosphate H
OH OH OH

Glycinamide Ribotide (GAR) Formylglycinamide ribotide (FGAR) Inosine Monophosphate (IMP)

BIOSINTESIS PURIN :
CO2 PERNAPASAN
6
SUMBER ATOM-ATOM C GLISIN
PURIN : 7
N
ASPARTAT
1C 5 C 8 N5N10METE-
N10-FORMIL- NIL TETRA-
C HIDRO FOLAT
TETRA-HIDRO-
FOLAT 2C 4 C 9
N
3 N
Amida dari GLUTAMIN

REAKSI SINTESIS PURIN :

1. PEMBENTUKAN FOSFORIBOSIL PIRO FOSFAT = PRPP
ATP AMP
RIBOSA-5-P 5-FOSFORIBOSIL-1-PIRO FOSFAT
PRPP - SINTASE
( PRPP )
PRPP JUGA PRAZAT UNTUK PIRIMIDIN, NAD dan NADP

2. PEMBENTUKAN 5-FOSFO- RIBOSILAMIN :

 GLUTAMIN GLUTAMAT
H20 PPi
PRPP 5-FOSFO- -RIBOSIL AMIN
PRPP-GLUTAMIN AMIDO TRANSFERASE

ATP AMP

3. 5-FOSFO- -RIBOSIL AMIN + GLISIN GLISIN AMIDA

GAR-SINTASE
RIBOSIL-5-FOSFAT ( GAR )
Metenil-tetra
hidro folat Tetra hidro folat
4. GLISIN AMIDA RIBO- FORMIL GLISIN AMI-
DA RIBOSIL FOSFAT (
SIL-5-FOSFAT (GAR) GAR FORMIL TRANS- FGAR )
FERASE
GLUTAMIN
GLUTAMAT
5. FORMIL GLISIN AMIDA FORMIL GLISIN AMI
DIN RIBOSIL FOSFAT
RIBOSIL FOSFAT (FGAR)
ATP ADP
H2 O
6. FORMIL GLISIN AMIDIN AMINO IMIDAZOL
RI-BOSIL-5-FOSFAT
RIBOSIL FOSFAT ATP ADP
 CO2
7. AMINO IMIDAZOL RIBO- AMINO IMIDAZOL KARBOK-
SIAMIDA RIBOSIL-5 P
SIL -5-FOSFAT
H2 O
8. AMINO IMIDAZOL KARBOK- AMINO IMIDAZOL SUK-
SINIL KARBOKSI RIBO-
SIAMIDA RIBOSIL-5 P
ASPARTAT SIL-5-P

9. AMINO IMIDAZOL SUKSI- AMINO IMIDAZOL KARBOK

SIAMIDA RIBOSIL-5-P
NIL KARBOKSI RIBOSIL-5-P
FUMARAT
N10Formil tetra Tetra hidro
hidro folat folat
10. AMINO IMIDAZOL KARBOK FORMIMIDO IMIDA-
ZOL ARBOKSIAMI-
SIAMIDA RIBOSIL-5-P
DA RIBOSIL-5-P
11. FORMIMIDO IMIDAZOL INOSIN MONO
KARBOKSIAMIDA RIBOSIL-5-P FOSFAT ( IMP )

SELANJUTNYA IMP DIUBAH MENJADI AMP dan GMP

 ADENILO SUKSINASE
IMP ADENILO SUKSINAT AMP
AMPS-SINTASE (ADENOSIN
NAD+ ( AMPS ) MONO
H2 O
PHOSPHAT )
IMP-DEHIDROGENASE
NADH +
GLUTAMAT
H+
GLUTAMIN
XANTOSIN MONO FOSFAT GUANOSIN MONO
ATP PHOSPHAT ( G M P )
( XMP )

SENYAWA / OBAT YANG DAPAT MENGHAMBAT SINTESIS

PURIN : . ANTI FOLAT REAKSI 4 ( TETRA HIDRO FOLAT )
. AZASERIN REAKSI 5
. DIAZANORLEUSIN REAKSI 2
. 6-MERKAPTOPURIN REAKSI 13 & 14
. ASAM MIKOFENOLAT REAKSI 14
 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 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 Reactions
Hydrolyzing a phosphate from ATP is relatively easy
G= -30.5 kJ/mol
If endergonic reaction released energy into cell as heat energy,
wouldnt be useful
Must be coupled to an exergonic 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; energase)
 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
 In Class Activity
***

Calculate how many ATP equivalents are needed for the de novo synthesize IMP.
Assume that all of the substrates (R5P, glutamine, etc) are available

Note: You should be able to do this calculation for the synthesis of

any of the nucleoside monophosphates
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 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 !
5-Phospho- -D-ribosyl-1-pyrophosphate
(PRPP)

Intermediet untuk baik proses de novo and

salvage pathway
Berasal dari ribosa 5 phosphat
 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
Biosintesis De Novo
Purines
 IMP synthase

GAR synthetase

AICAR
transformylase

GAR
transformylase

SAICAR lyase

FGAR amidotransferase

SAICAR synthetase
FGAM cyclase AIR karboksilase
Hal-hal penting dalam sintesis de novo
purin:

1. Sangat tergantung pada pool ribosa

2. Gugus amina didonor oleh glutamin dgn enzim
amidotransferase
3. Glisin dan fumarat donor ring dlm nukleotida
4. Daur reaksi dikontrol secara alosterik dgn AMP, ADP,
GMP dan GDP. bekerja pada
PRPP amidotransferase
Daur diawali dgn
perubahan PRPP IMP

IMP = Inosine monofosfat

mrpkn bentuk nukleotida
purin yang pertama
dibentuk dlm daur ini

Sebagai basa adalah

hypoxanthin
1. GLUTAMINE-PHOSPHORIBOSYLPYROPHOSPHATE
AMIDOTRANSFERASE

Glutamine
2. GLYCINAMIDE RIBONUCLEOTIDE
SYNTHETASE

H2O

Glycine
 3. GLYCINAMIDE RIBONUCLEOTIDE
FORMYLTRANSFERASE

Glycinamide ribotide Formylglycinamide ribotide

N10-formyltetrahydrofolate Tetrahydrofolate
4. PHOSPHORIBOSYLFORMYLGLYCINAMIDE
SYNTHETASE

Glutamine
5. AMINOIMIDAZOLE RIBONUCLEOTIDE SYNTHETASE
6. AMINOIMIDAZOLE RIBONUCLEOTIDE
CARBOXYLASE

CO2
7. SUCCINYLAMINOIMIDAZOLECARBOXAMIDE
RIBONUCLEOTIDE SYNTHETASE
8. ADENYLOSUCCINATE LYASE

Aspartate Fumarate
 9. 5-AMINOIMIDAZOLE-4-CARBOXAMIDE
RIBONUCLEOTIDE FORMYLTRANSFERASE

5-Aminoimidazole-4-carboxamide 5-Formaminoimidazole-4-
ribotide carboxamide ribotide

N10-formyltetrahydrofolate Tetrahydrofolate
10. IMP CYCLOHYDROLASE

H2O
Pengubahan IMP menjadi GMP
Pembentukan
guanilat (GMP) :
inosinat di oksidasi
menjadi Xantinat
dan C-2 pada gugus
carbonnya diganti
dengan gugus
amino,
membutuhkan ATP
 Adenilosuksinat
synthetase

IMP dehidrogenase

XMP aminase

Adenilosuksinat
lyase

DAUR dr IMP
AMP & GMP
BIOSINTESIS PIRIMIDIN :
PADA BIOSINTESIS PURIN dan PIRIMIDIN TERDAPAT BEBERAPA
PRECURSOR YANG SAMA, YAITU PRPP, GLUTAMIN,CO2 & ASPARTAT
REAKSINYA :
KARBAMOIL-P SINTASE II
1. CO2 + GLUTAMIN + ATP KARBAMOIL-P
ASPARTAT TRANSKARBAMOILASE
2. KARBAMOIL-P + ASPARTAT KARBAMOIL-ASPARTAT

DEHIDRO OROTASE
3. KARBAMOIL-ASPARTAT AS. DEHIDRO OROTAT
( DHOA )
DEHIDROGENASE
4. AS. DEHIDRO OROTAT + NAD+ ASAM OROTAT
OROTAT FOSFO RIBOSIL TRANSFERASE
5. ASAM OROTAT + PRPP OROTIDIN MONOFOSFAT
( OMP )
CO2
6. OROTIDIN MONOFOSFAT URIDIN MONOFOSFAT ( UMP )
OROTODILAT DEKARBOKSILASE
 ATP ADP ATP ADP GLUTAMIN

7. UMP UDP UTP CTP

CTP SINTASE
NADPH + H+ REDUKTASE

NADP+
H2 O Pi Metilen H4folat Folat
dUDP dUMP TMP
TIMIDILAT
SINTASE
 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 : Aspartate

C2 : HCO3-
N3 : Glutamine amide Nitrogen
 Pyrimidine Synthesis O

2 ATP + HCO3- + Glutamine + H2O C

HN CH
2 ADP +
Carbamoyl O
Glutamate +
Phosphate C C
Pi
Synthetase II
C O N
HN CH PRPP PPi COO
2-
O3P O CH2
O
NH2
C C Orotate Phosphoribosyl H H
N Transferase
O C O H H
H COO OH OH
O PO3-2 Orotidine-5'-monophosphate
Orotate
(OMP)
Carbamoyl Phosphate
Reduced
Aspartate Quinone OMP
Aspartate Dihydroorotate
Transcarbamoylase Decarboxylase
Dehydrogenase CO2
(ATCase)
Pi Quinone O

O C
O HN CH

C
HO C C CH
HN CH2 N
CH2 O
NH2 H2O
2-
C CH O3P O CH2
O
C CH O N H H
Dihydroorotase
O N H COO H H
H COO OH
OH
Dihydroorotate
Carbamoyl Aspartate Uridine Monophosphate
(UMP)
 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
 OMP DECARBOXYLASE : THE MOST
CATALYTICALLY PROFICIENT ENZYME
FINAL REACTION OF PYRIMIDINE PATHWAY
ANOTHER MECHANISM FOR DECARBOXYLATION
A HIGH ENERGY CARBANION INTERMEDIATE NOT
NEEDED
NO 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 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
 Sintesis de novo nukleotida pirimidine

CP synthetase

Aspartat
transcarbomoylase

Dihidrooratate DH
dihydrorotase
 Orotat
fosforibosiltransferas
e

Orotidilate
CTP synthetase dekarboksilase

UMP kinase
Nukleosida diphosphat
kinase
Sintetis karbamoil fosfat
 Sintetis karbamoil fosfat

Penyusun karbamoil fosfat dikatalis oleh sitosol carbomoil fosfat sintetase II (CPS II)
 Sintesis Asam Orotat
Tahap sintesis pirimidin
1.Pembentukan karbamoil aspartat (CA) dikatalisis oleh
aspartat transkarbamoilase
2.Cincin pirimidin kemudian tertutup secara hidrolitik oleh
Dihidroorotase sehingga menjadi Dihidroorotat
3.Dihidroorotat yang dihasilkan kemudian teroksidasi untuk
menghasilkan asam orotat, Enzim yang menghasilkan orotat
yakni dihidroorotat dehidrogenase
 Sintesis Asam Orotat
H2O

Pi

Aspartat

NAD

NADH + H
1.Di katalisi oleh Aspartat transkarbamoilase
2.Dihidroorotase
3.Teroksidasi ko enzim NAD, Dihidroorotat
dehidrogenase
 Pembentukan Nukleotida
Pirimidin

1. Orotat di ubah menjadi Oritidin 5 monofosfat (OMP)

2. PRPP menyumbangkan ribosa 5 Fosfatnya
3. Enzim orotat fosforibosil tranferase melepaskan pirofosfat
menghasilkan OMP
4. OMP yang merupakan induk dari mononeukletida pirimidin, akan
di ubah oleh orotidilat dekarbolase yang membuang gugus
karboksil yang bersifat asam menjadi Uridin monofosfat (UMP)
Pembentukan Nukleotida
Pirimidin
Pembentukan Nukleotida
Pirimidin
 Sintesis uridin trifosfat
Adanya Fosforilasi UMP kinase akan menjadikan UDP dan UTP
 Sintesis sitidin trifosfat

1. CTP terbentuk dari UTP

2. Penggantian gugus karbonil dengan gugus amino
 Sintesis Timin
1. UDP direduksi Ribonukleotida reduktase menjadi dUDP (deoksiuridin difosfat)
2. dUDP mengalami pelepasan Fosfat menghasilkan dUMP
3. N,N metilen Tetrahidrofolat memberikan gugus metilnya dengan bantuan Timidilat sintase membentuk dTMP
Sintesis Timidin monofosfat dari
dUMP
Hal-hal penting dalam sintesis de novo pirimidine:

cincin pirimidine disintesis terpisah dr gula ribosa nya

Daur pirimidine de novo tidak bercabang produk
akhir dr daur adalah UMP yang mrpkn bahan dari
CMP
Reaksi pertama pembtkan karbamoyl aspartate dr
asp dan carbomoyl-P titik regulasi yg penting dlm
daur tsb
Aspartat transcarbomoylase (ATCase) diaktivasi
oleh diaktivasi oleh ATP dan dihambat oleh CTP sbg
produk akhir
 KELAINAN METABOLISME PURIN
:
KATABOLISME NUKLEOSIDA PURIN ( ADENOSIN dan GUANOSIN )
PRODUK AKHIRNYA : ASAM URAT URINE
ASAM URAT : ASAM LEMAH & SUKAR LARUT dalam AIR; DALAM CAIR-
AN TUBUH DAPAT BERUPA GARAM ( Na URAT ).
GARAM Na URAT BERSIFAT LEBIH LARUT DARI PADA ASAM URAT,
YANG JUGA DIBUANG MELALUI URINE.
KRISTALISASI GARAM Na URAT DAPAT TERJADI DI :
-. GINJAL BATU
-. JARINGAN LUNAK & PERSENDIAN ENDAPAN
TOFUS INFLAMASI AKUT / KRONIS :
ARTHRITIS GOUT AKUT dan
ARTHRITIS GOUT KRONIS.
SELAIN PENYAKIT GOUT, JUGA TERDAPAT KELAINAN LAIN dari
METABOLISME PURIN : LIHAT TABEL
HIPERURISEMIA = KADAR ASAM URAT SERUM MELEBIHI BATAS
NORMAL.
 TABEL : KELAINAN BAWAAN PADA METABOLISME PURIN.
KELAINAN ENZIM YANG KARAKTERISTIK POLA PE-
KLINIS CACAT KELAINAN KLINIS WARISAN
GOUT PRPP SINTASE OVER PRODUKSI & OVER RESESIF -
SEKRESI PURIN TERKAIT X
GOUT PRPP SINTASE OVER PRODUKSI & OVER RESESIF -
SEKRESI PURIN TERKAIT X
GOUT PRPP SINTASE OVER PRODUKSI & OVER KEMUNGKIN-
SEKRESI PURIN AN RSESIF X

GOUT PRPP SINTASE OVER PRODUKSI & OVER RESESIF -

SEKRESI PURIN TERKAIT X

SINDROM LESCH HGPRT-ASE OVER PRODUKSI & OVER RESESIF -

-NYHAN SEKRESI PURIN ; SELF TERKAIT X
MUTA-TION
DEFISINSI IMUN ADENOSIN DE- IMUNO DOFISIENSI KOMBI- RESESIF -
AMINASE NASI ( SEL T & B ) AUTOSOMAL
NEFROLITIASIS ADENIN FOSFO NEFROLITIASIS 2,8-DEHI RESESIF -
RIBOSILTRANSF DROKSI ADENIN AUTOSOMAL
XANTINURIA XANTIN OKSI- NEFROLITIASIS XANTIN, RESESIF -
DASE HIPOXANTIN AUTOSOMAL
 A CASE STUDY : GOUT
A 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 : GOUT
THE 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.
 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
 ALCOHOL CONSUMPTION AND GOUT

Choi HK, Atkinson K, Karlson EW et al. . 2004. Alcohol intake and risk of incident gout in men:
a prospective study. Lancet 363: 1277-1281
Lesch-Nyhan Syndrome
A defect in production or activity of
HGPRT
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
 Purine Autism
25% of autistic patients may
overproduce purines
To diagnose, must test urine over
24 hours
Biochemical findings from this test
disappear in adolescence
Must obtain urine specimen in
infancy, but its difficult to do!
Pink urine due to uric acid crystals may
be seen in diapers
 IN-CLASS QUESTION
***
IN von GIERKES DISEASE, OVERPRO-
DUCTION OF URIC ACID OCCURS. THIS
DISEASE IS CAUSED BY A DEFICIENCY OF
GLUCOSE-6-PHOSPHATASE.

EXPLAIN THE BIOCHEMICAL EVENTS THAT LEAD

TO INCREASED URIC ACID PRODUCTION?
WHY DOES HYPOGLYCEMIA OCCUR IN THIS
DISEASE?
WHY IS THE LIVER ENLARGED?
 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?
 KELAINAN METABOLISME PIRIMIDIN :

HASIL METABOLISME PIRIMIDIN BERSIFAT SANGAT LARUT DALAM AIR

OVER PRODUKSI KATABOLIT PIRIMIDIN JARANG MENIMBULKAN

KELAINAN KLINIS

ASIDURIA ASAM OROTAT TIPE I :

-. DIFISAIENSI OROTAT FOSFORIBOSILTRANSFERASE
dan atau OROTIDILAT DEKARBOKSILASE

ASIDURIA ASAM OROTAT TIPE II :

-. DEFISIENSI OROTIDILAT DEKARBOKSILASE
-. LEBIH RINGAN DIBANDING DENGAN TIPE I
-. DAPAT DIATASI DENGAN PEMBERIAN URIDIN ORAL
NUKLEOTIDA ALAMIAH :
NUKLEOTIDA BEBAS YANG TIDAK TERIKAT DENGAN ASAM NUKLEAT;
BANYAK TERDAPAT DIJARINGAN TUBUH, a.l. :
DERIVAT ADENOSIN :
=. ADP = ADENOSIN DIFOSFAT
=. ATP = ADENOSIN TRIFOSFAT
=. c AMP = 31-51ADENOSIN MONOFOSFAT
ATP cAMP AMP
ADENILAT SIKLLASE FOSFO DIESTERASE
=. S-ADENOSIN METIONIN SEBAGAI DONOR METIL
DERIVAT GUANIN : GDP. GTP, GMP.
DERIVAT URASIL : -. URIDIN DIFOSFOGLUKOSA ( UDP-GLUKOSA )

-. URIDIN DIFOSFOGLUKORONAT ( UDP-GLUKORONAT

DERIVAT VITAMIN :.-.FLAVIN
UTP DINUKLEOTIDA = FAD
. NIKOTINAMID ADENIN DINUKLEOTIDA = NAD
. NIKOTINAMID ADENIN DINUKLEOTIDA FOSFAT = NAD
DERIVAT SINTETIK :

* 5-YODO-2-DEOKSI URIDIN
* 5-FLUORO MURASIL
* 6-MERKAPTO PURIN
* 6-TIOGUANIN
* AZAURIDIN
* AZAGUANIN
* 4- HIDROKSI PIRAZOL PIRIMIDIN
( ALOPURINOL )
 SH O
SH
N N N
N N N
N
H2N N H2N N
N N N
N H H
H
6-MERKAPTOPURIN 6- TIOGUANIN 8-AZAGUANIN

OH
O
N
F N
HN
N
N H
O
N
H ALLOPURINOL
5-FLUOROURASIL
 DNA vs. RNA: REVIEW
DNA composed of deoxyribonucleotides

Ribose sugar in DNA lacks hydroxyl group at 2

Carbon

Uracil doesnt (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 in vitro

 RIBONUCLEOTIDE REDUCTASE

R1 SUBUNIT
Three allosteric sites
Specificity Site
Hexamerization site
Activity Site
Five redox-active SH groups from cysteines

R2 SUBUNIT
Tyr 122 radical
Binuclear Fe(III) complex
 Ribonucleotide Reductase R2
Subunit

Fe prosthetic group binuclear, with each Fe

octahedrally coordinated
Fes are bridged by O-2 and carboxyl gp of Glu 115
Tyr 122 is close to the Fe(III) complex stabilization
of a tyrosyl free-radical
During the overall process, a pair of SH groups
provides the reducing equivalents
A protein disulfide group is formed
Gets reduced by two other sulfhydryl gps of Cys
residues in R1
 Chime Exercise
E. coli Ribonucleotide Reductase:

3R1R and 4R1R: R1 subunit

1RIB and 1AV8: R2 subunit

Explore 1AV8: Ribonucleotide Reductase in detail.This is the R2

subunit of E. coli Ribonucleotide Reductase. The biological molecule
consists of a heterotetramer of 2 R1 and two R2 chains.

Identify the following structures:

8 long -helices in one unit of R2

Tyr 122 residue
The binuclear Fe (III) complex
The ligands of the Fe (III) complex
 Mechanism of Ribonucleotide Reductase
Reaction
Free Radical
Involvement of multiple SH groups
RR is left with a disulfide group that must
be reduced to return to the original
enzyme
 RIBONUCLEOTIDE REDUCTASE

ACTIVITY IS RESPONSIVE TO LEVEL OF CELLULAR

NUCLEOTIDES:
ATP ACTIVATES REDUCTION OF
CDP
UDP
dTTP
INDUCES GDP REDUCTION
INHIBITS REDUCTION OF CDP. UDP
dATP INHIBITS REDUCTION OF ALL NUCLEOTIDES
dGTP
STIMULATES ADP REDUCTION
INHIBITS CDP,UDP,GDP REDUCTION
 RIBONUCLEOTIDE REDUCTASE

CATALYTIC ACTIVITY VARIES WITH STATE OF

OLIGOMERIZATION:
WHEN ATP, dATP, dGTP, dTTP BIND TO SPECIFICITY SITE
OF R1 (CATALYTICALLY INACTIVE MONOMER)
CATALYTICALLY ACTIVE (R1)2
WHEN dATP OR ATP BIND TO ACTIVITY SITE OF DIMERS
TETRAMER FORMATION
(R1)4a (ACTIVE STATE) == (R1)4b (INACTIVE)
WHEN ATP BINDS TO HEXAMERIZATION SITE
CATALYTICALLY ACTIVE HEXAMERS (R1)6
 Thioredoxin
Physiologic reducing agent of RNR
Cys pair can swap H atoms with disulfide formed
regenerate original enzyme
Thioredoxin gets oxidized to disulfide

Oxidized Thioredoxin gets reduced by NADPH ( final electron acceptor)

mediated by thioredoxin reductase
 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
 CHIME EXERCISE: dUTPase
1DUD: Deoxyuridine-5'-Nucleotide Hydrolase in a complex with
a bound substrate analog, Deoxyuridine-5'-Diphosphate
(dUDP).

Explore dUTPase as follows:

Find the substrate in its binding site

Find C5 on the Uracil group. Is there enough room to attach a
methyl group to C5?
Locate the ribose 2 C. What protein group sterically prevents an
OH group from being attached to the 2 C atom?
Find the H-bond donors and acceptors (to the uracil base) from the
protein. What would be the effect on the H-bonding if the base was
changed to cytosine?
 Tetrahydrofolate (THF)
Methylation of dUMP catalyzed by thymidylate
synthase
Cofactor: N5,N10-methylene THF
Oxidized to dihydrofolate
Only known rxn where net oxidation state of
THF changes
THF Regeneration:
DHF + NADPH + H+ THF + NADP+ (enzyme: dihydrofolate reductase)
THF + Serine N5,N10-methylene-THF + Glycine
(enzyme: serine hydroxymethyl transferase)
 REGENERATION OF N5,N10 METHYLENETETRAHYDROFOLATE

dUMP dTMP

thymidylate synthase

N5,N10 METHYLENE-THF DHF

NADPH + H+
GLYCINE
dihydrofolate reductase
serine hydroxymethyl
transferase
NADP+
SERINE
THF
 INHIBITORS OF N5,N10 METHYLENETETRAHYDROFOLATE
REGENERATION

dUMP dTMP

thymidylate synthase

N5,N10 METHYLENE-THF DHF

FdUMP NADPH + H+
GLYCINE
dihydrofolate reductase
serine hydroxymethyl
transferase
NADP+
SERINE X
THF
METHOTREXATE
AMINOPTERIN
TRIMETHOPRIM
 Anti-Folate Drugs
Cancer cells consume dTMP quickly for DNA
replication
Interfere 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
&