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Nucleotides metabolism
【目的与要求】
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•
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记住嘌呤核苷酸有两条合成途径。结合嘌呤核苷酸
结构与从头合成途径，说出嘌呤核苷酸各元素或组
件的材料来源。熟记二磷酸核苷还原生成脱氧嘌呤
核苷酸。写出与嘌呤核苷酸补救合成有关的酶的名
称、功能、酶缺陷相关的疾病
结合嘌呤核苷酸合成途径、调节，熟记嘌呤核苷酸
抗代谢药物作用机理及临床意义
记住嘌呤核苷酸体内分解代谢终产物-尿酸及其与医
学的关系
熟记嘧啶核苷酸从头合成的原料及合成调节。说出
嘧啶核苷酸补救合成所需的酶及其催化的反应。明
白嘧啶核苷酸抗代谢药物作用机理，记住嘧啶核苷
酸分解代谢产物名称
Outline
• 8.1 Purine metabolism
-8.1.1 The Biosynthesis of Purines
-8.1.2 Purine Salvage
-8.1.3 De-oxyribonucleotide Synthesis
-8.1.4 Purine Degradation
• 8.2 Pyrimidine metabolism
-8.2.1 Biosynthesis of Pyrimidines
-8.2.2 Pyrimidine Degradation
Nuclear acid
digestion
food
(stomach)
protein
nuclear acid (RNA and DNA) (intestine)
(phosphodiesterase) Endonucleases RNase
DNase
ribonucleotide mononucleotide Deoxyribonucleotide
mmol
umol
(phosphoesterase) Nucleotidase
Phosphate
nucleoside
nucleosidase
Uric acid
(purines)
β-ureidopropionate
( primidines)
base
excrete
Ribose or ribose-1-phosphate
(戊糖代谢)
Biological Roles of Nucleotides
• Monomeric units of nucleic acids *
• “ Energy currency”(ATP)
*
• Regulation of physiological processes
– Adenosine controls coronary(冠脉) blood flow
– cAMP and cGMP serve as signaling molecules
• Precursor function
- GTP to tetrahydrobiopternin
• Coenzyme components
- 5’-AMP in FAD/NAD+
• Activated intermediates: UDP-Glucose
• Allosteric effectors- regulate themselves and others
思考？
8.1.1 Nucleotide Biosynthesis
• For both purines and pyrimidines there are
two means of synthesis
- de novo (from bits and parts)
- salvage (recycle from pre-existing nucleosides,and
bases)
• Ribose generates energy, but purine and
pyrimidine rings do not
• Nucleotide synthesis pathways are good
targets for anti-cancer/antibacterial strategies
Bases/Nucleosides/Nucleotides
Base
Adenine
Base + Sugar=
Base + Sugar + Phosphate=
Nucleoside
Nucleotide
Deoxyadenosine
Deoxyadenosine
5’-triphosphate
(dATP)
The Pyrimidine Ring
The Purine Ring
Purine
Pyrimidine
De novo purine biosynthesis
• John Buchanan (1948) "traced" the sources of all
nine atoms of purine ring
1. In de novo synthesis, Inosine-5'-P (Inosine
Monophosphate, IMP) is the first nucleotide
formed
2. It is ,then, converted to either AMP or GMP
Location: liver cellular Cytoplasm
De novo purinenucleotide synthesis proceeds by the
synthesis of the purine base upon the ribose sugar moiety
•N-1: aspartic acid
•C-2:THF - one carbon units
•N-3: glutamine
•C-4, C-5, N-7: glycine
甘氨当中站, 谷氮坐两边,
•C-6: CO2
•C-8: THF - one carbon units 左上天冬氨, 头顶CO2
•N-9: glutamine
还有俩一碳
C-6
N-1
C-5
N-7
C-8
C-2
C-4
N-3
N-9
H
The metabolic origin of the nine atoms
in the purine ring system
1. First, synthesis Inosine-5'-P
(Inosine Monophosphate, IMP)
O
O
P O
HO
OH
H H
H H
ATP
HO
'
R-5 -P
OH OH
PRPP synthetase
O
HO
O
P O
OH
H H
HO
H H
OH OH
'
'
PP-1 -R-5 -P(PRPP)
_
P P
5-磷酸核糖胺,PRA
T1/2 30s
甘氨酰胺核苷酸(GAR)
甲酰甘氨酰胺核苷酸
(FGAR)
甲酰甘氨咪核苷酸(FGAM)
5-氨基咪唑核苷酸
(AIR)
5-氨基咪唑-4-羧酸核苷酸
5-氨基咪唑-4-(N-琥珀酸)
-甲酰胺核苷酸(SAICAR)
5-氨基咪唑-4-(N-琥珀酸)
-甲酰胺核苷酸(SAICAR)
5-氨基咪唑-4-甲酰胺
核苷酸(AICAR)
5-甲酰胺基咪唑4-甲酰胺核苷酸(FAICAR)
PRPP
Inosine
monophosphate
1 carbon via
folate
NH3 via
aspartylsuccinate
ATP dependent
step
(2) ATP
dependent step
NH3 via
glutamine
ATP dependent
step
1 carbon via
folate
ATP dependent
step
NH3 via
glutamine
ATP dependent
step
2. Second, Making AMP and GMP
kinase
AMP
ADP
ATP
GMP
ATP
ADP
kinase
GDP
ADP
kinase
ATP
ATP
ADP
kinase
ATP
GTP
ADP
Purines are
synthesized on
the Ribose ring
2.End product
inhibition and
“feed
forward”
regulation
ATP provides
the energy for
GMP synthesis
1. Committed Steps
( at the first two steps ):
PRPP , PRA
(A bunch of steps you
don’t need to know)
Regulation of De Novo Synthesis
3. “cross
regulation”
occurs from
IMP to AMP
and GMP
Feedback
Inhibition
GTP provides
the energy for
AMP synthesis
Committed Step
8.1.2 Salvage Pathway for Purines
Hypoxanthine
+ PRPP = IMP or GMP + PPi
or
Hypoxanthineguanosylphosphoribosyl transferase
Guanine
(HGPRTase)
Adenine
+ PRPP =
AMP + PPi
Adeninephosphoribosyl transferase
(APRTase)
Salvage pathways are particularly important in
brain/marrow that lack de novo purine synthesis
Lesch-Nyhan Syndrome(莱-尼综合症)
Absence of HGPRTase
X-linked (Gene on X)
Occurs primarily in males
Characterized by:
purine synthesis is increased
200-fold
Increased uric acid
Spasticity(痉挛)
Neurological defects
Aggressive behavior
Self-mutilation(自残)
Inter-conversion of Purine nucleotides
GMP
AMP
NH3
AMPS
（腺苷酸代琥珀酸）
IMP
XMP
8.1.3 Deoxyribonucleotide Biosynthesis
BASE
HOCH
OH
2
O
5´
H H 1´
4´
H 3´
HO
HOCH2 O BASE
OH
5´
H 1´
4´ H
H 3´ 2´ H
Ribonucleotide HO
OH
H
2´
H
Reductase
Deoxyribonucleoside
Ribonucleoside
ribonucleotide reductase
NDP
dNDP
ribonucleotide reductase
ADP
dADP
ribonucleotide reductase
GDP
dGDP
ribonucleotide reductase
UDP
dUDP
ribonucleotide reductase
CDP
TDP
dCDP
dTDP
Deoxyribonucleotide Biosynthesis ?
硫氧还蛋白
Mg2+
Ribonucleotides can be converted to deoxyribonucleotides
by Ribonucleotide Reductase at the diphosphate level
E. coli
Ribonucleotide Reductase
Regulates
the level of
cellular
dNTPs
The ribonucleotide reductase, An (R1)2(R2)2- type
enzyme , has R1 (86 kD) and R2 (43.5 kD) two subunits
dNDP+ATP
kinase
dNTP+ADP
dADP+ATP
kinase
dGDP+ATP
kinase
dUDP+ATP
kinase
dUTP+ADP
kinase
dCTP+ADP
dCDP+ATP
phosphorylase
dNDP
dTTP
?
dATP +ADP
dGTP+ADP
dNMP+Pi
Regulation of dNTP Synthesis
• The overall activity of ribonucleotide reductase
must be regulated
• Balance of the four deoxynucleotides must be
controlled
• ATP activates, dATP inhibits at the overall activity
site
• ATP, dATP, dTTP and dGTP bind at the specificity
site to regulate the selection of substrates and the
products made
Tumor
over-growth + Heterogeneity
( nucleotides + protein )
How to inhibit the biosynthesis of the tumor cells?
for anti-cancer strategies(antibacterial)
Chemotherapeutic Agents
1. Analogs of purine:
OH
SH
N
N
N
N
N
N
H
SH
OH
N
N
N
N
H
6-巯基鸟嘌呤
N （6-mercaptopurine,
6-MP）
H
N
N
N
H2N
inosine
6-巯基嘌呤
N
N
N
H
(6-mercaptoguanine)
8-氮杂鸟嘌呤
(8-azoguanine)
2. Analogs of amino acids:
O
NH2
H2N—C—CH2—CH2—CH—COOH
Gln
O
Inhibit the reactions
of the Gln
NH2
N+ —N—CH2—C—O—CH2—CH—COOH
氮杂丝氨酸（azaserine）
O
NH2
N+ —N—CH2—C—CH2—CH2—CH—COOH
6-重氮-5-氧正亮氨酸(diazonnorleucine)
3. Analogs of Folic acid
NH2
N
N
H2N
N
N
H2N
N
H
H
N
R
O
COOH
H
O
COOH
H
—CH2—N—
—C—N—CH
CH2
R=H，aminopterin,氨喋呤
CH2
R=CH3，methotrexate,
COOH
氨甲喋呤,MTX
—CH2—N—
N
C
N
OH H
四氢叶酸,THF
H
—C—N—CH
CH2
CH2
COOH
PRPP
6MP
MTX
GAR
FGAR
PRA
Gln
azaserine
氮杂丝氨酸(azaserine)
FGAM
PPi
6MP
MTX
AICAR
PRPP
A
AMP
PPi
FAICAR
PRPP
IMP
6MP
The mechanism of the
Chemotherapeutic Agents
I
G
GMP
azaserine
PPi
PRPP
6MP
8.1.4 Purine catabolism
AMP
I
XO
X
GMP
XO
G
Sequential removal of bits and pieces
End product is uric acid
XO: Xanthine Oxidase
Excreted in
Urine
Xanthine Oxidase and Gout
＞0.48mmol/L(8mg%),
The scale of uric acid
(normal value) :
0.12～0.36mmol/L;
male, 0.27mmol/L;
formale, 0.21mmol/L
Allopurinol, which inhibits XO, is a treatment of gout
别嘌呤醇
次黄嘌呤
The mechanism of allopurinol as a treatment of gout
OH
OH
H
N
C
N
N
allopurinol
CH
N
N
N
N
N
H
H
PRPP ↓
I
XO
Allopurinol nucleotide
X
Purine
nucleotides
Uric acids
8.2 Pyrimidine Biosynthesis
Pyrimidine Biosynthesis:
In contrast to purines, First, synthesis of
the pyrimidine ring; Next, attachment of
ribose-phosphate to the ring
De Novo Pyrimidine Biosynthesis
HOOC
H2C
HC NH2
HOOC
Carbamoyl-P
Aspartate
The metabolic origin of the six atoms
of the pyrimidine ring
乳清酸核苷酸
乳清酸
二氢乳清酸
CTP From UTP
at the triphosphate level
kinase
kinase
UMP
ATP
UDP
ADP
ATP
UTP
ADP
Synthesis of Thymine Nucleotides
1. Thymine nucleotides are made from
dUMP, which derives from dUDP, dCDP
2. Biosynthesis of deoxyribonucleotides
by ribonucleotide reductase
3. Biosynthesis of thymidine monophosphate (dTMP)
by thymidylate synthase
O
C
HN
O
C
N
Thymidylate synthase methylates
dUMP at 5-position to make dTMP
CH
CH
dTMP synthase
HN
N5,N10- methylene FH4
dR-5'-P
FH4
dTMP
ATP
FH2
DHFR
NADP+
dUMP
O
C
O
C
NADPH+H
N
C-CH3
CH
'
dR-5
-P
+
dTMP
N5,N10-methylene
THF is 1-C donor
kinase
kinase
dTTP
dTDP
ADP
ATP
ADP
Regulation of Pyrimidine Synthesis（de novo）
• Aspartate transcarbamoylase (ATCase，细菌)
catalyzes the condensation of carbamoyl
phosphate with aspartate to form carbamoylaspartate
• Note that carbamoyl phosphate represents an
‘activated’ carbamoyl group
Feedback Inhibition
Regulation of Pyrimidines Biosynthesis
Regulation occurs at first step in the pathway (committed step)
×
2ATP + CO2 + Glutamine = carbamoyl phosphate
Inhibited by UTP
If you have lots of UTP around this means you won’t
make more that you don’t need
CPS II
• Carbamoyl phosphate for pyrimidine
synthesis is made by carbamoyl phosphate
synthetase II (CPS II，哺乳动物细胞)
• This is a cytosolic enzyme (whereas CPS I
is mitochondrial and used for the urea cycle)
• Substrates are HCO3-, glutamine, 2 ATP
Allosteric regulation of pyrimidine
biosynthesis
Enzyme regulated
carbamoyl
phosphate
synthetase II
Allosteric effector
UDP, UTP
PRPP, ATP
Effect
Feedback inhibition
stimulatory
CPS-I vs. CPS-II
?
Biosynthesis: Purine vs. Pyrimidine
start with ribose, build on
nitrogen base
Regulated by GTP/ATP
Generates IMP
Requires Energy
build nitrogen base then added
to PRPP
Synthesized Regulated by UTP
Generates UMP/CMP
Requires Energy
“Both are very complicated multi-step process which
your kindly professor does not expect you to know in detail”
Salvaging Pyrimidines
• Pyrimidines+PRPP  Nucleoside+PPi
(嘧啶磷酸核糖转移酶)
• A second type of salvage pathway involves two steps
and is the major pathway for the pyrimidines, uracil
and thymine
Base + Ribose 1-phosphate = Nucleoside + Pi
(nucleoside phosphorylase)
• Nucleoside + ATP  Nucleotide + ADP
(nucleoside kinase-irreversible)
Inhibitors of pymidines synthesis are cancer drugs
Analogs of pymidines /pymidine nucleosides:
N
O
F
HN
O
N
H
5-氟尿嘧啶,
5-Fu
NH·HCl
C
C
C
O
N
O
HOH2C
H
NH2
C
N
C
C
O
H
HO H
OH
H
阿糖胞苷
Cytarabine
H
C
N
O
HOH2C
H
OH
C
H
H
环胞苷
Cyclocytidine
氮杂丝氨酸 azaserine
阿糖胞苷Cytarabine
UMP
UTP
CTP
CDP
dCDP
MTX
UDP
dUDP
dUMP
dTMP
5FU
(5FdUMP/5FUTP)
Pyrimidine Catabolism-1
NH2
C
N
CH
CH
C
N
O
H
O
NADPH+H+ NADP+
C
NH3
HN
CH
HN
CH
C
C
N
O
O
H
C
β-Alanine
U
CO2+NH3
H2N-CH2-CH2-COOH
O
C
N
H
CH2
CH2
H2O
O
H2O HO C
H2N
CH2
CH2
C
N
O
H
O
C
HN
O
C
N
H
NADPH+H+
C-CH3
CH
O
C
NADP+
HN
O
C
N
H
CH-CH3
CH2
DHT
T
H2O
Pyrimidine Catabolism-2
CO2+NH3
H2N-CH2-CH-COOH
CH3
β-氨基异丁酸
β-aminoisobutyrate
O
H2O HO C
H2N
CH-CH3
CH2
C
N
O
H
β-脲基异丁酸
'
5 -P-R
PRPP
IMP
CO2+Gln
H2N-CO-P
overview
OMP
AMP dAMP dGMP GMP UMP dUMP CMP dCMP dTMP
ADP dADP dGDP GDP UDP dUDP CDP dCDP dTDP
dUTP
ATP
dATP
dGTP GTP UTP
CTP
De novo synthesis
dCTP dTTP
CPS-I vs. CPS-II
氨基甲酰磷酸合成酶I
(CPS-I)
分布
线粒体(肝)
氮源
氨
氨基甲酰磷酸合成酶II
(CPS-II)
胞液
谷氨酰胺
变构激活剂
N-乙酰谷氨酸
无
变构抑制剂
无
UMP
功能
尿素合成
嘧啶合成