Zhihong Li （李志红）Department of Biochemistry

Biochemistry Ⅱ

Main TopicsMetabolism of Nucleotides (4h)

DNA replication; RNA transcription; Protein synthesis (12h)

Gene expression and regulation (4h); Recombinant DNA technology (4h)

Signal transduction; Oncogene (6h)

Diabetes mellitus (2h); Lipoproteins Metabolism (4h)

Cholesterol Metabolism (2h); Bile acids Metabolism (2h)

Examination

Heme Synthesis (2h); Bile Pigments Metabolism (2h)

Liver function tests (2h); Metabolism of xenobiotics (2h)

Plasma Proteins and Immuno Proteins (2h)

Free Radicals and Antioxidants (2h)

Mineral Metabolism:(micro-elements) (2h)

Water and Electrolyte Balance; Acid Base Balance (4h)

Hormones (6h)

Biochemical changes during Pregnancy (2h)

N

N

N

NH

N

N

Lecture 1

Metabolism of Nucleotides

Contents

• Review: Structure of nucleic acid• Degradation of nucleic acid• Synthesis of Purine Nucleotides• Degradation of Purine Nucleotides• Synthesis of Pyrimidine Nucleotides• Degradation of Pyrimidine Nucleotides

Nucleoside and Nucleotide

Nitrogenous base ribose

Nitrogenous base ribose phosphate

Nucleoside =

Nucleotide =

pyrimidine purineOR

Riboseor

2-deoxyribose

N--glycosylbond

Structure of nucleotides

Purines vs Pyrimidines

Section 1

Degradation of nucleic acid

Nucleoprotein

Nucleic acid Protein

Nucleotide

NucleosidePhosphate

Base Ribose

Nucleotidase

Nucleosidase

Degradation of nucleic acid

In stomach Gastric acid and pepsin

In small intestine Endonucleases: RNase and DNase

Significances of nucleotides

1. Precursors for DNA and RNA synthesis

2. Essential carriers of chemical energy, especially ATP

3. Components of the cofactors NAD+, FAD, and coenzyme A

4. Formation of activated intermediates such as UDP-glucose and CDP-diacylglycerol.

5. cAMP and cGMP, are also cellular second messengers.

Section 2

Synthesis of Purine Nucleotides

There are two pathways leading to nucleotides

• De novo synthesis: The synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose-5-phosphate, CO2, and one-carbon units.

• Salvage pathways: The synthesis of nucleotide by recycle the free bases or nucleosides released from nucleic acid breakdown.

§ 2.1 De novo synthesis• Site:

– in cytosol of liver, small intestine and thymus

• Characteristics:

a. Purines are synthesized using 5-phosphoribose(R-5-P) as the starting material step by step.

b. PRPP(5-phosphoribosyl-1-pyrophosphate) is active donor of R-5-P.

c. AMP and GMP are synthesized further at the base of IMP(Inosine-5'-Monophosphate).

N10-Formyltetrahydrofolate

N10-Formyltetrahydrofolate

1. Element sources of purine bases

First, synthesis Inosine-5'-Monophosphate, IMP

2. Synthesis of Inosine Monophosphate (IMP)

• Basic pathway for biosynthesis of purine ribonucleotides

• Starts from ribose-5-phosphate(R-5-P)• Requires 11 steps overall• occurs primarily in the liver

OH

1 ATP

AMP

2

Gln:PRPP amidotransferase

ribose phosphate pyrophosphokinase

Step 1:Activation of ribose-5-phosphate

Step 2: acquisition of purine atom N9

5- 磷酸核糖胺 ,PRA

•Steps 1 and 2 are tightly regulated by feedback inhibition

Committed step

甘氨酰胺核苷酸

3

Step 3: acquisition of purine atoms C4, C5, and N7

glycinamide synthetase

甲酰甘氨酰胺核苷酸

4

•Step 4: acquisition of purine atom C8

GAR transformylase

FH4 (or THF)

N10—CHO—FH4

甲酰甘氨咪核苷酸

5

Step 5: acquisition of purine atom N3

6

5- 氨基咪唑核苷酸

•Step 6: closing of the imidazole ring

5- 氨基 -4- 羧基咪唑核苷酸

Carboxyaminoimidazole

ribonucleotide (CAIR)

7

Step 7: acquisition of C6

AIR carboxylase

5- 氨基 -4-(N- 琥珀酸 )

- 甲酰胺咪唑核苷酸

Carboxyaminoimidazole ribonucleotide (CAIR)

Step 8: acquisition of N1

SAICAR synthetase

5- 氨基 -4- 甲酰胺咪唑核苷酸

Step 9: elimination of fumarate

adenylosuccinate lyase

5- 甲酰胺基 -4- 甲酰胺咪唑核苷酸

Step 10: acquisition of C2

AICAR transformylase

Step 11: ring closure to form IMP

• Once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells).

N10-CHOFH4

N10-CHOFH4

3. Conversion of IMP to AMP and GMP

Note: GTP is used for AMP synthesis.

Note: ATP is used for GMP synthesis.

IMP is the precursor for both AMP and GMP.

kinase

ADPkinase

ADP

ATP

ATP ADP

AMP

ATP

kinase

GDPkinase

ADP

GTP

ATP ADP

GMP

ATP

4. ADP, ATP, GDP and GTP biosynthesis

5. Regulation of de novo synthesis

The significance of regulation:

(1) Fulfill the need of the body, without wasting.

(2) [GTP]=[ATP]

• Purine nucleotide biosynthesis is regulated by feedback inhibition

§ 2.2 Salvage pathway

• Purine bases created by degradation of RNA or DNA and intermediate of purine synthesis were costly for the cell to make, so there are pathways to recover these bases in the form of nucleotides.

• The significance of salvage pathway :– a. Save the fuel.– b. Some tissues and organs such as brain and bone marr

ow are only capable of synthesizing nucleotides by salvage pathway.

• Two phosphoribosyl transferases are involved:– APRT (adenine phosphoribosyl transferase) for adenine.– HGPRT (hypoxanthine guanine phosphoribosyl transferas

e) for guanine or hypoxanthine.

Purine Salvage Pathway

N

NN

N

NH2

O

Guanine

N

N N

O

N

Hypoxanthine

O

OHHO

2-O3POH2C

N

N N

O

N

IMP

O

OHHO

2-O3POH2C

N

NN

N

NH2

O

GMP

.

.

Adenine AMP

PRPP PPi

adenine phosphoribosyl transferase

PRPP PPi

hypoxanthine-guaninephosphoribosyl transferase

(HGPRT)

Absence of activity of HGPRT leads to Lesch-Nyhan syndrome.

Lesch-Nyhan syndrome

• first described in 1964 by Michael Lesch and William L. Nyhan.

• there is a defect or lack in the HGPRT enzyme• Sex-linked metabolic disorder: only males• the rate of purine synthesis is increased about 200-fold

– Loss of HGPRT leads to elevated PRPP levels and stimulation of de novo purine synthesis.

• uric acid level rises and there is gout• in addition there are mental aberrations• patients will self-mutilate by biting lips and fingers off

Lesch-Nyhan syndrome

§ 2. 3 Formation of deoxyribonucleotide

• Formation of deoxyribonucleotide involves the reduction of the sugar moiety of ribonucleoside diphosphates (ADP, GDP, CDP or UDP).

• Deoxyribonucleotide synthesis at the nucleoside diphosphate(NDP) level.

SS

H2OMg2+

NADPH + H+NADP+

SHSH

thioredoxin

ribonucleotide reductase

NDP£¨N=A, G, C, U£©

dNDP

dNTP

ATP

ADP

kinase

O BaseCH2

HOH

OP PO BaseCH2

OHOH

OP P

thioredoxin

thioredoxin reductase

FAD

Deoxyribonucleotide synthesis at the NDP level

§ 2. 4 Antimetabolites of purine nucleotides

• Antimetabolites of purine nucleotides are structural analogs of purine, amino acids and folic acid.

• They can interfere, inhibit or block synthesis pathway of purine nucleotides and further block synthesis of RNA, DNA, and proteins. Widely used to control cancer.

1. Purine analogs

• 6-Mercaptopurine (6-MP) is a analog of hypoxanthine.

N

N NH

N

OH

N

N NH

N

SH

6-MPhypoxanthine

6-MP 6-MP nucleotide

de novo synthesis

salvage pathway

HGPRT

amidotransferase

IMP

AMP and GMP

--

-

-

-

• 6-MP nucleotide is a analog of IMP

2. Amino acid analogs

• Azaserine (AS) is a analog of Gln.

H2N C CH2

O

CH2 CH

NH2

COOH Gln

C

O

CH2 CH

NH2

COOH ASNN CH2 O

3. Folic acid analogs

• Aminopterin (AP) and Methotrexate (MTX)

R＝H：AP

folic acid

N

NN

N

NH2

H2N

CH2 N C

R O

NH CH

COOH

R＝CH3：TXT

CH2 CH2 COOH

N

NN

N

OH

H2N

CH2 N C

H O

NH CH

COOH

CH2 CH2 COOH

MTX

folate FH2 FH4

NADPH + H+

NADP+NADPH + H+

NADP+

FH2 reductase FH2 reductase

AP or MTX

- -

•The structural analogs of folic acid(e.g. MTX) are widely used to control cancer (e.g. leukaemia).

•Notice: These inhibitors also affect the proliferation of normally growing cells. This causes many side-effects including anemia, baldness, scaly skin etc.

Section 3

Degradation of Purine Nucleotides

nucleotide

nucleotidaseH2O

Pi

nucleosidenucleoside

phosphorylase

Pi R-1-P

purine

R-5-P

PRPP

pentose phosphate pathway

salvage pathway

oxidationuric acid

N

HCN

C

CC

N

CH

N

NH2

Ribose-P

AMP

HN

HCN

C

CC

N

CH

N

O

Ribose-P

IMPHN

HCN

C

CC

NH

CH

N

O

HN

CNH

C

CC

NH

CH

N

O

O

HN

CNH

C

CC

NH

C

N

O

O

O

GMP

Hypoxanthine

Uric Acid Xanthine

Xanthine Oxidase

N

HCN

C

CC

N

CH

N

NH2

Ribose-P

AMP

HN

HCN

C

CC

N

CH

N

O

Ribose-P

IMPHN

HCN

C

CC

NH

CH

N

O

HN

CNH

C

CC

NH

CH

N

O

O

HN

CNH

C

CC

NH

C

N

O

O

O

GMP

Hypoxanthine

Uric Acid Xanthine

Xanthine Oxidase

(2,6,8-trioxypurine)

Adenosine Deaminase

• Uric acid is the excreted end product of

purine catabolism in primates, birds, and some other animals.

• The rate of uric acid excretion by the normal adult human is about 0.6 g/24 h, arising in part from ingested purines and in part from the turnover of the purine nucleotides of nucleic acids.

Uric acid

• The disease gout, is a disease of the joints, usually in males, caused by an elevated concentration of uric acid in the blood and tissues.

• The joints become inflamed, painful, and arthritic, owing to the abnormal deposition of crystals of sodium urate.

• The kidneys are also affected, because excess uric acid is deposited in the kidney tubules.

GOUT

The uric acid and the gout

Uric acid Over 8mg/dl, in the plasma

Gout, Urate crystallization in joints, soft tissue, cartilage and kidney

Hypoxanthine

Xanthine Out of body

In urine

Diabetese nephrosis

……

Advanced GoutClinically Apparent Tophi

1

1. Photos courtesy of Brian Mandell, MD, PhD, Cleveland Clinic.2. Photo courtesy of N. Lawrence Edwards, MD, University of Florida.3. ACR Clinical Slide Collection on the Rheumatic Diseases, 1998.

21

3

HN

HCN

C

CC

NH

CH

N

O

Hypoxanthine

HN

HCN

C

CC

NH

N

HC

O

Allopurinol

Allopurinol – a suicide inhibitor used to treat Gout

Xanthine oxidase

Xanthine oxidase

Adenosine Deaminase(ADA) and Severe Combined Immunodeficiency Disease (SCID)

• SCID is caused by an Adenosine Deaminase Deficiency (ADA)– Gene is located on chromosome #22– ADA is involved in purine degradation – Accumulation of nucleotide metabolites = TOXIC t

o developing T lymphocytes – Deficiency results in failure to develop functional T

and B lymphocytes– Patients cannot withstand infection die if untrea

ted

Boy in the Bubble

Gene therapy

• September 14, 1990 @ NIH, French Anderson and R. Michael Blaese perform the first GT Trial– Ashanti (4 year old girl)

• Her lymphocytes were gene-altered (~109) used as a vehicle for gene introduction using a retrovirus vector to carry ADA gene

– Cynthia (9 year old girl) treated in same year

Section 4

Synthesis of Pyrimidine Nucleotides

• shorter pathway than for purines• Pyrimidine ring is made first, then attached to ribo

se-P (unlike purine biosynthesis)• only 2 precursors (aspartate and glutamine, plus H

CO3-) contribute to the 6-membered ring

• requires 6 steps (instead of 11 for purine)• the product is UMP (uridine monophosphate)

§ 4.1 De novo synthesis

1. Element source of pyrimidine base

N

CN

C

CC

12

34

5

6Asp

CO2

Gln

•Carbamoyl phosphate synthetase(CPS) exists in 2 types: •CPS-I, a mitochondrial enzyme, is dedicated to the urea cycle and arginine biosynthesis. •CPS-II, a cytosolic enzyme, used here. It is the committed step in animals.

Step 1: synthesis of carbamoyl phosphate

Step 2: synthesis of carbamoyl aspartate

ATCase: aspartate transcarbamoylase

•Carbamoyl phosphate is an “activated” compound, so no energy input is needed at this step.

Step 3: ring closure to form dihydroo

rotate

Step 4: oxidation of dihydroorotate to orotate

Step 5: acquisition of ribose phosphate moiety

•ribose phosphate originates from PRPP

Step 6: decarboxylation of OMP

The big picture

3. UTP and CTP biosynthesis

UDP

ADP

UTP

ATP ADP

UMP

ATP

kinase kinase

4. Formation of dTMP

The immediate precursor of thymidylate (dTMP) is dUMP.

The formation of dUMP either by deamination of dCMP or by hydrolyzation of dUDP. The former is the main route.

dTMP dTDP dTTP

dUMP

dUDP dCMP dCDP

N5,N10-methylene-tetrahydrofolic Acid

ATP ATP

ADP ADP

dTMP synthetase

UDP

dTMP synthesis at the nucleoside monophosphate level.

dUMP

dUDP

dCMPdTMP

H2O

Pi

H2O

NH3

NADPH

NADP+

thymidylate synthase HN

N

O

O

R 5' Pd

CH3

reductase

HN

N

O

O

R 5' Pd

+ H+

FH2

FH4

N5, N10-CH2-FH4 FH2

5. Regulation of de novo synthesis

carbamoyl phosphate

carbamoyl aspartate

UMP

ATP + CO2 + Gln

PRPP

UTP CTP

ATP + R-5-P

purine nucleotide

pyrimidine nucleotide

§ 4. 2 Salvage pathway

+ ATP

+ ATP

+ ATP

UMPCMP

dTMP + ADP

dCMP + ADP

uridinecytidine

deoxythymidine

deoxycytidine

thymidine kinase

deoxycytidine kinase

uridine-cytidine kinase+ ADP

+ PRPP + PPiuracilthymineorotic acid

pyrimidine phosphate ribosyltransferase UMP

dTMPOMP

§ 4. 3 Antimetabolites of pyrimidine nucleotides

• Antimetabolites of pyrimidine nucleotides are similar with them of purine nucleotides.

1. Pyrimidine analogs

• 5-fluorouracil (5-FU) is a analog of thymine.

HN

NH

O

O

FHN

NH

O

O

CH3

thymine5-FU

2. Amino acid analogs

• Azaserine (AS) inhibits the synthesis of CTP.

3. Folic acid analogs

• Methotrexate (MTX) inhibits the synthesis of dTMP.

4. Nucleoside analogs

• Arabinosyl cytosine (ara-c) inhibits the synthesis of dCDP.

N

N

NH2

O

ara-c

O

H

OH H

H

CH2OH

H OH

N

N

NH2

O

cytosine

O

H

OH OH

H

CH2OH

H H

Section 5

Degradation of Pyrimidine Nucleotides

H2OH2O

H2N CH2 CH2 COOH H2N CH2 CH COOH

CH3

N

NH

O

NH2H2O NH3

HN

NH

O

O

CH2

CH2NH2

NH

O

HOOC

HN

NH

O

O

CH3

CH2

CHNH2

NH

O

HOOC

CH3

cytosine uracil thymine

¦Â-ureidopropionate

¦Â-ureido-isobutyrate

CO2 + NH3

¦Â-alanine ¦Â-aminoisobutyrate

Highly soluble products

Summary of purine biosynthesis

　dATP

dGTP

AMP

GMP

ADP

GDP

dADP

dGDP

IMP

ATP

GTP

CTP

Summary of pyrimidine biosynthesis

　

UDP UTP

CDP

dUDP

dCDP

dUMP

dCMP

dTMP

UMP

dTDP dTTP

dCTP

Summary of Nucleotide Synthesis

• Purines built up on ribose– PRPP synthetase: key step– First, synthesis IMP

• Pyrimidine rings built, then ribose added– CPS-II: key step– First, synthesis UMP

• Salvage is important

Points• Synthesis of Purine Nucleotides

– De novo synthesis: Site, Characteristics, Element sources of purine bases

– Salvage pathway: definition, significance, enzyme, Lesch-Nyhan syndrome

– Formation of deoxyribonucleotide: NDP level– Antimetabolites of purine nucleotides:

• Purine, Amino acid, and Folic acid analogs

• Degradation of Purine Nucleotides– Uric acid, gout

• Synthesis of Pyrimidine Nucleotides– De novo synthesis: Characteristics, Element sources of pyri

midine bases– Salvage pathway– Antimetabolites of pyrimidine nucleotides

• Catabolism of Pyrimidine Nucleotides