Nucleotides metabolism

【目的与要求】• 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

• 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

Outline

Deoxyribonucleotide umol

food

protein nuclear acid (RNA and DNA) (intestine)

Endonucleases(phosphodiesterase)

mononucleotide

Nucleotidase(phosphoesterase)

nucleosidePhosphate

nucleosidase

Ribose or ribose-1-phosphatebase

Nuclear acid digestion

Uric acid (purines)

β-ureidopropionate( primidines)

RNase

DNase

(stomach)

ribonucleotide mmol

( 戊糖代谢 )excrete

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

BaseBase + Sugar=

NucleosideBase + Sugar + Phosphate=

NucleotideAdenine 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 ni

ne 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 CytoplasmCytoplasm

De novo purinenucleotide synthesis proceeds by the synth

esis of the purine base upon the ribose sugar moiety

The metabolic origin of the nine atoms The metabolic origin of the nine atoms in the purine ring systemin the purine ring system

•N-1: aspartic acid •C-2:THF - one carbon units•N-3: glutamine •C-4, C-5, N-7: glycine •C-6: CO2 •C-8: THF - one carbon units•N-9: glutamine

N-1

C-2

N-3

C-6

C-8

N-9

H

甘氨当中站 , 谷氮坐两边 ,

左上天冬氨 , 头顶 CO2

还有俩一碳

N-7

C-4

C-5

1. First, synthesis Inosine-5'-P

(Inosine Monophosphate, IMP)

R-5'-P

PRPP synthetase

PP-1'-R-5'-P(PRPP)

OH

O

OH P O

OH

H

OH

H

OH

HH

O

OH

O

OH P O

OH

H

OH

H

OH

HH

O

ATP

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)

NH3 via glutamine

NH3 via glutamine

1 carbon via folate

1 carbon via folate

NH3 via aspartyl- succinate

(2) ATP dependent step

ATP dependent step

ATP dependent step

ATP dependent step

ATP dependent step

PRPP

Inosine monophosphate

2. Second, Making AMP and GMP

kinaseADP

kinase

ADP

ATP

ATP ADP

AMPATP

kinaseGDP

kinase

ADP

GTP

ATP ADP

GMPATP

Regulation of De Novo Synthesis

ATP provides the energy for GMP synthesis

GTP provides the energy for AMP synthesis

3. “cross regulation” occurs from IMP to AMP and GMP

2.End product inhibition and “feed forward” regulation

1. Committed Steps( at the first two steps ):

PRPP , PRA(A bunch of steps you don’t need to know)

Purines are synthesized on the Ribose ring

FeedbackInhibition

Committed Step

8.1.2 Salvage Pathway for Purines

Hypoxanthineor

Guanine

+ PRPP = IMP or GMP + PPi Hypoxanthineguanosylphosphoribosyl transferase

(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 HGPRTaseX-linked (Gene on X)

Occurs primarily in malesCharacterized by:

purine synthesis is increased 200-foldIncreased uric acidSpasticity( 痉挛 )Neurological defectsAggressive behaviorSelf-mutilation( 自残 )

AMP GMP

XMP AMPS（腺苷酸代琥珀酸）

IMP

NH3

Adenine DeaminaseNADP

+NH 3

NADPH

Guanine Red

uctase

Inter-conversion of Purine nucleotides

OH

HHO

H

H

HOCH2 OH

H

1´

2´3´

4´

5´BASE

OH

HHO

H

H

HOCH2

OH

OH

1´

2´3´

4´5´

BASE

Deoxyribonucleoside Ribonucleoside

RibonucleotideReductase

8.1.3 Deoxyribonucleotide Biosynthesis

NDP dNDPribonucleotide reductase

ADP dADP

ribonucleotide reductase

GDP dGDP

ribonucleotide reductase

UDP dUDP

ribonucleotide reductase

CDP dCDP

ribonucleotide reductase

TDP dTDP

Deoxyribonucleotide Biosynthesis ?

Mg2+硫氧还蛋白

Ribonucleotides can be converted to deoxyribonucleotides

by Ribonucleotide Reductase at the diphosphate level

The ribonucleotide reductase, An (R1)2(R2)2- type enzyme

, has R1 (86 kD) and R2 (43.5 kD) two subunits

E. coli Ribonucleotide Reductase

Regulates the level of

cellular dNTPs

dNDP+ATP dNTP+ADPkinase

kinasedCDP+ATP dCTP+ADP

dUDP+ATP dUTP+ADPkinase

dGDP+ATP dGTP+ADPkinase

dADP+ATP dATP +ADPkinase

dTTP ?

dNDP dNMP+Piphosphorylase

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

over-growth + Heterogeneity

( nucleotides + protein )

Tumor

How to inhibit the biosynthesis of the tumor cells?

for anti-cancer strategies(antibacterial)

Chemotherapeutic Agents1. Analogs of purine:

8- 氮杂鸟嘌呤

N

OH

N

NNH

N

SH

N

NNH

N

SH

N

NNH

H2N

N

OH

N

NNH

N

6- 巯基鸟嘌呤

inosine6- 巯基嘌呤

（ 6-mercaptopurine,

6-MP ）

(8-azoguanine)

(6-mercaptoguanine)

2. Analogs of amino acids:

H2N—C—CH2—CH2—CH—COOH

O NH2

N+ —N—CH2—C—O—CH2—CH—COOH

O NH2

N+ —N—CH2—C—CH2—CH2—CH—COOH

O NH2

Gln

氮杂丝氨酸（ azaserine ）

6- 重氮 -5- 氧正亮氨酸 (diazonnorleucine)

Inhibit the reactions

of the Gln

3. Analogs of Folic acid

R=H ， aminopterin, 氨喋呤R=CH3 ， methotrexate,

氨甲喋呤 ,MTX

N

NH2

N

NNH

H2N

—CH2—N—

R

—C—N—CH

O

CH2

CH2

COOH

COOHH

N

NCH

—CH2—N—

H

—C—N—CH

O

CH2

CH2

COOH

COOHH

H2N

N

OH

NH

四氢叶酸 ,THF

PRPP

Gln

6MP

氮杂丝氨酸 (azaserine)

PRA GAR FGAR

FGAM

MTX

azaserine

AICAR

MTXFAICAR IMP

AMP

GMP

PRPPPPi

PPi PRPP

6MP

6MP

6MPazaserine

A

IG

PRPPPPiThe mechanism of the Chemotherapeutic Agents

AMP I

GMP G

XXO

XO

Excreted in

Urine

Sequential removal of bits and pieces

End product is uric acid

XO: Xanthine Oxidase

8.1.4 Purine catabolismcatabolism

The scale of uric acid (normal value) :

0.12 ～ 0.36mmol/L;

male, 0.27mmol/L;

formale, 0.21mmol/L

＞ 0.48mmol/L(8mg%),

Xanthine Oxidase and Gout

别嘌呤醇 次黄嘌呤

Allopurinol, which inhibits XO, is a treatment of gout

C

OH

N

NNH

H

NN

OH

N

NNH

CH

I

allopurinol

PRPP

Allopurinol nucleotideXO

Purine nucleotides

The mechanism of allopurinol as a treatment of gout

↓

Uric acids

X

8.2 Pyrimidine Biosynthesis

Pyrimidine Biosynthesis:

In contrast to purines, First, synthesis of

the pyrimidine ring; Next, attachment of ribos

e-phosphate to the ring

Carbamoyl-PAspartate

De Novo Pyrimidine Biosynthesis

The metabolic origin of the six atoms The metabolic origin of the six atoms of the pyrimidine ringof the pyrimidine ring

NH2HCH2C

HOOC

HOOC

乳清酸 二氢乳清酸乳清酸核苷酸

CTP From UTP at the triphosphate level

UDPADP

UTP

ATP ADP

UMPATP

kinase kinase

1. Thymine nucleotides are made from dU

MP, which derives from dUDP, dCDP

2. Biosynthesis of deoxyribonucleotides b

y ribonucleotide reductase

3. Biosynthesis of thymidine monophosphate (dTMP)

by thymidylate synthase

Synthesis of Thymine Nucleotides

CO

HNC

CH

CHN

O

dR-5'-P

dUMP

CO

HNC

C-CH3

CHN

O

dR-5'-P

dTMP synthase

FH2N5,N10- methylene FH4

FH4

DHFR

NADPH+H+NADP+

dTMP

kinasedTDP

kinase

ADP

dTTP

ATP ADP

dTMPATP

Thymidylate synthase methylates dUMP at 5-position to make dTMP

N5,N10-methylene THF is 1-C donor

Regulation of Pyrimidine Synthesis （ de novo ）

• Aspartate transcarbamoylase (ATCase ，细菌 ) catalyzes the condensation of carbamoyl

phosphate with aspartate to form carbamoyl-a

spartate

• 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)

Inhibited by UTPIf you have lots of UTP around this means you won’t

make more that you don’t need

×2ATP + CO2 + Glutamine = carbamoyl phosphate

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 Allosteric effector Effect

carbamoyl phosphate synthetase II

UDP, UTP Feedback inhibition

PRPP, ATP stimulatory

CPS-I vs. CPS-II ?

Biosynthesis: Purine vs. Pyrimidine

start with ribose, build on nitrogen baseRegulated by GTP/ATPGenerates IMPRequires Energy

build nitrogen base then added to PRPPSynthesized Regulated by UTPGenerates UMP/CMPRequires Energy

“Both are very complicated multi-step process whichyour 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 a

nd 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)

Analogs of pymidines /pymidine nucleosides:

5- 氟尿嘧啶 ,5-Fu

阿糖胞苷 Cytarabine

环胞苷Cyclocytidine

N

NH

O

O

F

HHOH2C

H

HHO

OH

HH

O

C

C

C

N

N

CO

NH2

HOH2C

H

H

OH

HH

O

C

C

C

N

N

C

NH·HCl

O

Inhibitors of pymidines synthesis are cancer drugs

UMP

UDP

UTP

dUDP dUMP dTMP

CTP CDP dCDP

MTX

5FU(5FdUMP/5FUTP)

阿糖胞苷 Cytarabine

氮杂丝氨酸 azaserine

CO

NC

CH

CHN

NH2

HC

O

HNC

CH

CHN

O

H

NH3

CO

HNC

CH2

CH2N

O

H

NADPH+H+ NADP+

H2O

CO

H2NC

CH2

CH2N

O

H

HOH2OCO2+NH3

H2N-CH2-CH2-COOH

β-Alanine

C U

Pyrimidine Catabolism-1

CO

HNC

C-CH3

CHN

O

H

NADPH+H+ NADP+

CO

HNC

CH-CH3

CH2N

O

H

CO

H2NC

CH-CH3

CH2N

O

H

HOH2OCO2+NH3

β-aminoisobutyrate

H2N-CH2-CH-COOH

CH3

β- 脲基异丁酸

H2OT DHT

Pyrimidine Catabolism-2

β- 氨基异丁酸

overview5'-P-R

PRPP

IMP

dAMP GMPdGMPAMP

dADP GDPdGDPADP

dATP GTPdGTPATP

UMP CMPdUMP

UDP CDPdUDP

UTP CTP

dUTP

dTMPdCMP

dTDPdCDP

dTTPdCTP

CO2+Gln

H2N-CO-P

OMP

De novo synthesis

dUDP

dCMPdUMP

CPS-I vs. CPS-II

氨基甲酰磷酸合成酶 I 氨基甲酰磷酸合成酶 II

分布 线粒体 (肝 ) 胞液

氮源 氨 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

(CPS-I) (CPS-II)