Nucleotide Metabolism

Nucleotide MetabolismNucleotide Metabolism

Nucleic acid metabolism

DNA metabolismDNA metabolism

RNA metabolismRNA metabolism

主要内容 :

核酸的降解及代谢

DNA的复制与修复

RNA的生物合成

核酸代谢

• Energy metabolism (ATP)*

• Monomeric units of nucleic acids*

• Regulation of physiological processes – Adenosine controls coronary 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 othe

rs

Cellular Roles of Nucleotides

How I hope to make this at least bearable if not mildly interesting• Purines and Pyrimidines

– Synthesis (de novo and salvage pathways)

– Degradation

– Relevant disease states

– Relevant clinical applications

You are not responsible for any structures

Purines and Pyrimidines

Adenine Guanine

Thymine/Uracil Cytosine

Two Purines

Two Pyrimidines

HN

CHN

C

CN

CN

C

NH2

H

NC

CC

HN

C

O

CH3

HO

HN

C

CC

N

CH

O

H

H

NH2

HN

CHN

C

CN

CN

C

O

H2N

H

Nucleotide MetabolismNucleotide Metabolism

Nucleotide degradationNucleotide degradation

Nucleotide synthesisNucleotide synthesis

Nucleotide degradationNucleotide degradation

Purine Degradation

Pyrimidine Degradation

核苷酸酶（磷酸单酯酶）

专一性的磷酸单酯酶 : 3ˊ- 核苷酸酶 , 5ˊ- 核苷酸酶

非专一性磷酸单酯酶

核苷酸酶 核苷酸磷酸化酶核苷酸 核苷 碱基 + （脱氧）戊糖磷酸

磷酸解

水解

核苷 + H2O 核苷水解酶

碱基 + 核糖

核苷 + Pi 核苷磷酸化酶

碱基 + 核糖 -1-P

Purine Degradation

• Sequential removal of bits and pieces

• End product is uric acid • Uric acid is primate-specific

Other species further metabolize

uric acid

Excreted inUrine

Xanthine Oxidase

( 黄嘌呤 )

( 尿酸 )

灵长类的动物

Pyrimidine Degradation

• Pyrimindine rings can be fully degraded to soluble structures (Compare to purines that make uric acid)

Degradation pathways are quite distinct for purines and pyrimidines, but salvage pathways are quite similar

Purine Degradation

• Sequential removal of bits and pieces

• End product is uric acid • Uric acid is primate-specific

Other species further metabolize

uric acid

Excreted inUrine

Xanthine Oxidase

灵长类的动物

不同种类的生物分解嘌呤的能力不同，产物也不同。人、灵长类、鸟类、某些爬虫类将嘌呤分解成尿酸，其他生物还可将尿酸进一步分解成尿囊素、尿囊酸、

尿素、甚至 CO2 、 NH3 。

嘌呤的分解代谢 :

核酸中的嘌呤主要是 Ade 、 Gua 首先脱氨，分

别生成次黄嘌呤和黄嘌呤，再进一步代谢生成尿酸。

Inosine: 次黄嘌呤核苷Hypoxanthine: 次黄嘌呤Xanthine: 黄嘌呤Uric acid: 尿酸Allantoin: 尿囊素Allantoic acid: 尿囊酸

尿囊酸

尿囊素

尿酸

Inosine: 次黄嘌呤核苷Hypoxanthine: 次黄嘌呤Xanthine: 黄嘌呤Uric acid: 尿酸

软骨鱼

灵长类鸟类爬行类昆虫类

其它孵乳类

硬骨鱼类

两栖动物

Uric acid: 尿酸；Allantoin: 尿囊素；Allantoic acid: 尿囊酸

Uric acid: 尿酸；Allantoin: 尿囊素；Allantoic acid: 尿囊酸

Purine catabolism Purine catabolism in animalsin animals

Excess Uric Acid Causes Gout

• Primary gout (hyperuricemia，高尿酸血症 ) – Inborn errors of metabolism that lead to overproduction

of Uric Acid • Overactive de novo synthesis pathway

– Leads to deposits of Uric Acid in the joints – Causes acute arthritic joint inflammation

Offal foods such as liver, kidneys, tripe, sweetbreads and tongueAvoid:

Xanthine Oxidase

Allopurinol

X

( 别嘌呤醇 )

（痛风）

尿酸过多导致痛风（ gout ）

结构与次黄嘌呤很相似的别嘌呤醇（ allopurinol ）对黄嘌呤氧化酶有很强的抑制作用，可用来治疗痛风。

别嘌呤醇

IMP: 次黄嘌呤苷一磷酸

HGPRT: 次黄嘌呤苷鸟嘌呤磷酸核糖转移酶

别嘌呤醇

Immunodeficiency Diseases Associated with Purine Degradation

• Defect in adenosine deaminase – Removes amine from adenosine

• SCID- severe combined immunodeficiency

• “Bubble Boy” Disease • Defect in both B-cells and T-cell

s (Disease of Lymphocytes) • Patients extremely susceptible to

infection - hence the Bubble

Lymphocyte

Therapies for SCID

• Can be diagnosed in infants through a simple blood test (white cell count)

• Bone marrow transplant for infants – Familial donor

• Continued administration of adenosine deaminase (ADA-PEG)

• Gene therapy- repair defective gene in T-cells or blood stem cells

Pyrimidine Degradation

• Pyrimindine rings can be fully degraded to soluble structures (Compare to purines that make uric acid)

Degradation pathways are quite distinct for purines and pyrimidines, but salvage pathways are quite similar

RNA ： Cyt 、 Ura

嘧啶碱的分解代谢

DNA ： Thy

二氢尿嘧啶脱氢酶　

二氢嘧啶酶　

β-脲基丙酸酶　

甲基丙二酸半醛　

( 二氢胸腺嘧啶 )

二氢尿嘧啶脱氢酶　

(β- 脲基异丁酸 )

二氢嘧啶酶　

(β- 氨基异丁酸 )

(β- 脲基异丁酸 )

β-脲基丙酸酶　

(β- 氨基异丁酸 )

(甲基丙二酸半醛 )

Antimetabolites

• Often drugs that inhibit cell growth are used to combat cancer. Many of these compounds are analogues of purine and pyrimidine bases or nucleotides. Many of these drugs must be activated by cellular enzymes. They affect nucleic acid synthesis and tumor cells tend to be more susceptible since they are dividing more rapidly

6-Mercaptopurine (6-MP)

• Purine Analogue

PRPP + 6-MP 6-mercaptopurine ribonucleotide

Inhibitor of Committed Step in de novo Purine Biosynthesis

This reaction is more active in tumor cells

• Used clinically to combat childhood leukemia Since 1963 cure rate has increased from ~4% to greater than 80%

Cytosine Arabinose (araC)• Metabolized to cytosine arabinose 5’-triphosphate (ara

CTP) • Analogue of CTP • Incorporated into DNA and inhibits chain synthesis • Used extensively for acute leukemias

Cytosine Arabinose

Differs only in the sugar

NC

CC

N

CH

O

H

NH2

HOH

H

H

HO

OH

HOCH2N

C

CC

N

CH

O

H

NH2

HOCH2

OH

OHH

H

O

H

Cytosine Ribose

AZT as an Anti-HIV Agent

• 3’-Azido-2’-deoxythymidine • Pyrimidine Analogue • HIV is a retrovirus

• RNA genome that is reverse-transcribed to DNA Viral polymerase is inhibited by AZT

NC

CC

HN

C

O

HO

H

H

H

HO

HN3

CH3

HOCH2

AZT

AZT ： 3’- 叠氮 -2’- 脱氧胸腺嘧啶核苷

Antifolates

• Antifolates interfere with formation of dihydrofolate which is required for: – dTMP synthesis

– de novo purine biosynthesis

Thymidylate Synthase

DihydrofolateN5,N10-Methylene tetrahydrofolate

Tetrahydrofolate

Dihydrofolate ReductaseX

Antifolate Agents Mimic Folate

Hydroxyurea

• Specifically inhibits ribonucleotide reductase

• Inhibits DNA synthesis without affecting RNA synthesis or other nucleotide pools Cleared from the body rapidly so not used extensively in the clinic

H2N C

O

NHOH

The BIG Picture

• GMP, AMP, UMP on…..

• Generation of dTMP

• Common features of clinically relevant antimetabolites/antifolates Antiviral agents- how are they specific for the virally infected cells?

Nucleotide MetabolismNucleotide Metabolism

Nucleotide synthesisNucleotide synthesis

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

ynthesis (often regulate one another)

–de novo (from bits and parts)

–salvage (recycle from pre-existing nucleotides)

Salvage Pathway

5’

Many Steps Require an Activated Ribose Sugar (PRPP)

de novo Synthesis

• Committed step: This is the point of no return – Occurs early in the biosynthetic pathway – Often regulated by final product (feedback

inhibition)

X

Purine Biosynthesis (de novo)

1

23 4

56

7

8

9

Purine Biosynthesis (de novo)

• Atoms derived from: – Aspartic acid – Glycine – Glutamine – CO2

– Tetrahydrofolate

• Also requires – 4 ATP’s

Purines are synthesized on the Ribose ring

Committed Step

Inhibited by AMP, GMP, IMP X

Purine Biosynthesis (de novo)

ATP GTP

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

(Inosine Monophosphate)NCH

NC

CN

CN

C

O

H

NCH

NC

CN

CN

C

O

H2N

H

NCH

NC

CN

CN

C

NH2

HFeedback Inhibition

次黄嘌呤核苷一磷酸

The pathway fThe pathway for purine biosyor purine biosynthesisnthesis

(Gln-PRPP 氨基转移酶 )

( 甘氨酰核糖核苷酸 )

(GAR 转甲酰基酶 )

( 甲酰甘氨酰核糖核苷酸 )

( 甘氨酰核糖核苷酸 , GAR)

( 甲酰甘氨酰胺核糖核苷酸 )

( 甲酰甘氨酰核糖核苷酸 )

(5- 氨基咪唑核糖核苷酸 )

( 甲酰甘氨酰胺核糖核苷酸 )

(5- 氨基咪唑核糖核苷酸 )

(5- 甲酰氨基咪唑核糖核苷酸 )

( 甲酰氨基咪唑核糖核苷酸 )

( 甲酰氨基咪唑核糖核苷酸 )

(N- 琥珀酰 -5- 氨基咪唑 -4- 甲酰胺基核糖核苷酸 )

(N- 琥珀酰 -5- 氨基咪唑 -4- 甲酰胺基核糖核苷酸 )

(5- 氨基咪唑 -4- 甲酰胺基核糖核苷酸 )

(5- 氨基咪唑 -4- 甲酰胺基核糖核苷酸 )

(N- 甲酰氨基咪唑 -4- 甲酰胺基核糖核苷酸 )

(N- 甲酰氨基咪唑 -4- 甲酰胺基核糖核苷酸 )

次黄嘌呤核苷一磷酸

The synthesis of AMP and GMP from IMPThe synthesis of AMP and GMP from IMP

The metabolic origin of the nine atoms in the pThe metabolic origin of the nine atoms in the purine ring systemurine ring system

Salvage Pathway for Purines

Hypoxanthine or

Guanine

+ PRPP = IMP or GMP + PPi Hypoxanthineguanosylphosphoribosyl transferase

(HGPRTase)

Adenine + PRPP = AMP + PPi Adeninephosphoribosyl transfe

rase (APRTase)

(B) phosphoribosyl transferase

(A) Pu 嘌呤核苷 嘌呤核苷酸核苷磷酸化酶 核苷磷酸激酶

R-1-P Pi ATP ADP

嘌呤补救途径（ salvage pathway of purine ）

APRT AMP+PPi+PRPP腺嘌呤腺嘌呤磷酸核糖转移酶

IMP+PPi+PRPP次黄嘌呤-次黄嘌呤 鸟嘌呤

(HGPRT)磷酸核糖转移酶

-次黄嘌呤 鸟嘌呤(HGPRT)磷酸核糖转移酶+PRPP鸟嘌呤 GMP+PPi

(adenine phosohoribosyl transferase)

(hypoxanthine-guanine phosohoribosyl transferase)

• 节约能量和一些氨基酸的消耗。

• 有些组织（如脑、骨髓）不能从头合成嘌呤核苷酸，只能进行嘌呤核苷酸的补救合成。

– HGPRT 完全缺失的患儿，表现为自毁容貌综合症。

嘌呤核苷酸补救合成的生理意义

Lesch-Nyhan Syndrome

• Absence of HGPRTase • X-linked (Gene on X)

– Occurs primarily in males

• Characterized by: – Increased uric acid

– Spasticity

– Neurological defects

– Aggressive behavior

– Self-mutilation

Biosynthesis of Pyrimidines

• Pyrimidine rings are synthesized independent of the ribose and transferred to the PRPP (ribose)

• Generated as UMP (uridine 5’-monophosphate)

• Synthesized from: – Glutamine

– CO2

– Aspartic acid – Requires ATP

NC

CC

HN

C

O

CH3

HO

H

NC

CC

N

CH

O

H

H

NH2

Uracil Cytosine

The pyrimidine biosynthetic pathwayThe pyrimidine biosynthetic pathway

( 乳清酸 )

( 二氢乳清酸 )

( 乳清苷 -5’-P)

( 氨甲酰天冬氨酸 )

( 氨甲酰磷酸 )

Biosynthesis of Pyrimidines

( 乳清酸 )

( 二氢乳清酸 )

( 乳清酸苷 -5’-P)

( 氨甲酰天冬氨酸 )

( 天冬氨酸转氨甲酰基酶 )

( 二氢乳清酸酶 )

( 二氢乳清酸脱氢酶 )

( 乳清酸磷酸核糖基转移酶 )

( 乳清苷酸脱羧酶 )

( 胞苷酸合成酶 )

( 天冬氨酸转氨甲酰基酶 )

( 二氢乳清酸酶 )

( 二氢乳清酸脱氢酶 )

( 乳清酸磷酸核糖基转移酶 )

( 氨甲酰天冬氨酸 )

( 二氢乳清酸 )

( 乳清酸 )

( 乳清酸苷 -5’-P)

( 乳清苷酸脱羧酶 )

( 胞苷酸合成酶 )

Regulation of Pyrimidine 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

X

Hereditary Orotic Aciduria

• Defect in de novo synthesis of pyrimidines

• Loss of functional UMP synthetase C Gene located on chromosome III

• Characterized by excretion of orotic acid

• Results in severe anemia and growth retardation

• Extremely rare (15 cases worldwide)

• Treated by feeding UMP

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

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

Why does UMP Cure Orotic Aciduria?

Carbamoyl Phosphate Orotate

UMP Synthetase

X

Feedback Inhibition

Pyrimidine Salvage

• Can also be salvaged by reactions with PRPP

• -Catalyzed by Pyrimidine phosphoribosyltransferase

• Nucleoside kinase

Degradation pathways are quite distinct for purines and pyrimidines, but salvage pathways are quite similar

尿苷磷酸化酶Ura

R-1-P Pi

U （尿苷）

尿苷激酶

UMP

ATP

ADP

尿嘧啶磷酸核糖转移酶 Ura

PRPP PPi

嘧啶补救途径 (salvage pathway of pyrimidine)

补救途径

Cyt + PRPP CMP + PPi

胞苷激酶 CMPCATP ADP

( 胞苷 )

Biosynthesis: Purine vs Pyrimidine

• Synthesized on PRPP • Regulated by GTP/ATP • Generates IMP • Requires Energy

• Synthesized then added to PRPP

• 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

Wait a minute:So far we’ve only made GMP, AMP, and UMP

Beyond AMP, GMP and UMP

Purine Biosynthesis Pyrimidine Biosynthesis

But other forms of these nucleotides are needed

Two Problems

• These are monophosphates (i.e. GMP)- we need triphosphates (i.e. GTP) for both DNA and RNA synthesis These are ribonucleotides- that’s fine for RNA but we also need to make DNA

Synthesis of ribonucleotides first supports the RNA world theory

Synthesis of UTP/CTP (Easy Problem)

Nucleotide Diphosphokinase

ATP ATP

NC

CC

N

CH

O

H

H

NH2

NC

CC

HN

C

O

CH3

HO

H

NC

CC

HN

C

O

CH3

HO

H

ATP + Glutamine

UMP CMP + NH3

UMP

ATP ADP

UMP¼¤Ã¸UDP

ATP ADP

UDP¼¤Ã¸UTP

(1)¶¯Ö²Îï

(2)΢ ÉúÎï CTP

ADP ATP

CDPCDP¼¤Ã¸

CMP

ADP ATP

CMP¼¤Ã¸

Mg2+Mg2+

Mg2+Mg2+

胞苷酸（ CMP ）的合成

CTP synthesis from UTPCTP synthesis from UTP

Beyond dGTP, dATP and dUTPBeyond dGTP, dATP and dUTP

So far we’ve made GTP, ATP, and UTP for incorporation into RNA Also dGTP and dATP for incorporation into DNA

We still need dCTP for both RNA and DNA We also need to generate dTTP for DNA

Specific Kinases Convert NMP to NDP

Nucleoside Monophosphates

Nucleoside Diphosphates

Monophosphate Kinases

• Monophosphate kinases are specific for the bases

Adenylate Kinase

Guanylate KinaseGMP + ATP GDP + ADP

AMP + ATP 2ADP

Conversion of Ribonucleotides to Deoxyribonucleotides

OH

HHO

H

H

HOCH2

OH

OH

1´

2´3´

4´5´O

H

HHO

H

H

HOCH2 OH

H

1´

2´3´

4´

5´BASE BASE

Deoxyribonucleoside Ribonucleoside

Somehow we need to get rid of this damn oxygen

Ribonucleotide Reductase

Ribonucleotide ReductaseRibonucleotide Reductase

Catalyzes conversion of NDP to dNDP Highly regulated enzyme Regulates the level of cellular dNTPs Activated prior to DNA synthesis Controlled by feedback inhibition

E.coliE.coli ribonucleotide reductase ribonucleotide reductase

The free radical mechanism of ribonucleotide The free radical mechanism of ribonucleotide reductionreduction

Electron transfer from NADPH to RRElectron transfer from NADPH to RR

Ribonucleotide reductase- enzyme organizRibonucleotide reductase- enzyme organization and regulationation and regulation

activity specificity

Regulation of ribonucleotide redRegulation of ribonucleotide reductaseuctase

Cell Cycle [late G1] Allosteric regulation

– Overall activity: + ATP, -dATP – Substrate specificity:

• ATP stimulate CDP,UDP reduction • (d)TTP stimulates GDP reduction • (d)TTP inhibits CDP,UDP reduction • dGTP stimulates ADP reduction, inhibits GDP, CD

P, UDP reduction

DNA synthesDNA synthesisis

Synthesis of deoxSynthesis of deoxyribo-nucleotideyribo-nucleotides --- reduction at s --- reduction at the 2’-position of the 2’-position of the ribose ring of the ribose ring of nucleoside diphonucleoside diphosphatessphates

dNDP to dNTP (the final step)dNDP to dNTP (the final step)

Once dNDPs are generated by ribonucleotide reductase a general kinase can phosphorylate to make the dNTP’s

So far we’ve made (d) GTP, (d)ATP, and (d)CTP

What about TTP?

Synthesis of TTP(Hard Problem)

NC

CC

HN

C

O

CH3

HO

H

NC

CC

HN

C

O

CH3

HO

H

Thymidylate Synthase

• Methyl group is provided by N5,N10-Methylene tetrahydrofolate Dihyrofolate reductase recharges the Dihydrofolate to N5,N10-Methylene tetrahydrofolate

CH3

Role of Folate in dTMP Synthesis

Dihydrofolate Reductase

Thymidylate Synthase

DihydrofolateN5,N10-Methylene tetrahydrofolate

Tetrahydrofolate

SummarySummary

>90% of purines are salvaged Most de novo synthesis in liver, highly regu

lated Cross regulation of purine and pyrimidine n

ucleotide biosynthesis assures balanced levels of these metabolites

Disruption of salvage or catabolism leads to disease