Biochemistry

Zhihong Li
Department of Biochemistry

Main Topics
Metabolism of Nucleotides (4h)
DNA replication(4h); RNA transcription(4h); Protein synthesis (4h)
Gene expression and regulation (4h); Recombinant DNA technology (4h)
Signal transduction(4h); Oncogene(2h); Gene and disease (2h)
Diabetes mellitus (2h); Lipoproteins Metabolism (4h)
Cholesterol Metabolism (2h); Bile acids Metabolism (2h)
Plasma Proteins and Immuno Proteins (2h)
Inter-assesment
Free Radicals and Antioxidants (2h) ; Mineral Metabolism(2h)
Water and Electrolyte Balance(2h); Acid Base Balance (2h)
Heme Synthesis (2h); Bile Pigments Metabolism (2h)
Liver function tests (2h); Metabolism of xenobiotics (2h)
Hormones (6h); Biochemical changes during Pregnancy (2h)
Biochemistry of Cerebrospinal fluid(CSF)(2h)

Lecture 1
Metabolism of Nucleotides
N

N
N

N
H

N
N

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

Nucleoside = Nitrogenous base ribose
Nucleotide = Nitrogenous base ribose phosphate

Purines vs Pyrimidines

Structure of nucleotides
pyrimidine

OR

purine

N--glycosyl
bond
Ribose
or
2-deoxyribose

Section 1
Degradation of nucleic acid

Degradation of nucleic acid

Nucleoprotein
In stomach

Gastric acid and pepsin

Nucleic acid
In small intestine

Protein

Endonucleases: RNase and DNase

Nucleotide
Nucleotidase

Phosphate

Nucleoside
Nucleosidase

Base

Ribose

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 5phosphoribose(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).

1. Element sources of purine bases

N10Formyltetrahydrofolate

N10Formyltetrahydrofolate

First, synthesis Inosine-5'-Monophosphate, IMP

FH4 (or THF)

N10CHOFH4

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

Step 1:Activation of ribose-5-phosphate

Committed step

ATP
AMP

ribose phosphate pyrophosphokinase

Step 2: acquisition of purine atom N9

Gln:PRPP amidotransferase
Steps 1 and 2 are tightly
regulated by feedback inhibition

5- ,PRA

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

glycinamide synthetase

Step 4: acquisition of purine atom C8

4
GAR transformylase

Step 5: acquisition of purine atom N3

Step 6: closing of the imidazole ring

5-

Step 7: acquisition of C6

7
AIR carboxylase

Carboxyaminoimidazole
ribonucleotide (CAIR)
5- -4-

Step 8: acquisition of N1

Carboxyaminoimidazole
ribonucleotide (CAIR)

SAICAR synthetase

5- -4-(N- )
-

Step 9: elimination of fumarate

adenylosuccinate lyase

5- -4-

Step 10: acquisition of C2

AICAR transformylase

5- -4-

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.

4. ADP, ATP, GDP and GTP biosynthesis

kinase

kinase

AMP

ATP

ADP
ADP

ATP

ATP

kinase

ADP

kinase

GTP

GDP

GMP
ATP

ADP

ATP

ADP

5. Regulation of de novo synthesis

The significance of regulation:
(1) Meet the need of the body, without
wasting.
(2) AMP and GMP control their respective
synthesis from IMP by a feedback
mechanism, [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 can be
directly converted to the corresponding
nucleotides.
The significance of salvage pathway :
Save the fuel.
Some tissues and organs such as brain and bone marrow
are only capable of synthesizing nucleotides by salvage
pathway.

Two phosphoribosyl transferases are involved:

APRT (adenine phosphoribosyl transferase) for adenine.
HGPRT (hypoxanthine guanine phosphoribosyl
transferase) for guanine or hypoxanthine.

Purine Salvage Pathway

adenine
phosphoribosyl transferase

Adenine

PRPP

AMP

PPi
O
N

O
N

2-O

N
N
Hypoxanthine
O
N
N

hypoxanthine-guanine
phosphoribosyl transferase
(HGPRT)
PRPP

N
N

Guanine

NH2

3POH2C

HO OH
IMP

PPi

N
2-O

3POH2C

N
N

NH2

HO OH
GMP

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.

O CH2 O

Base

ribonucleotide
reductase

O CH2 O

Mg2+

Base

H 2O
OH H
S
SH
thioredoxin
thioredoxin
dNDP
NDP
S
SH
ATP
N=A, G, C, U
kinase
FAD
+
+
NADPH
+
H
NADP
thioredoxin
ADP
reductase
dNTP
OH OH

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 DNA, RNA, and proteins.
Widely used to control cancer.

1. Purine analogs
6-Mercaptopurine (6-MP) is a analog of
hypoxanthine.

 6-MP nucleotide is a analog of IMP

de novo synthesis

amidotransferase

6-MP

IMP

6-MP nucleotide

AMP and GMP

HGPRT

salvage pathway

2. Amino acid analogs

Azaserine (AS) is a analog of Gln.

3. Folic acid analogs

Aminopterin (AP) and Methotrexate (MTX)

MTX

NADPH + H+

NADP+

folate
FH2 reductase

NADPH + H+
FH2

NADP+

FH2 reductase

FH4

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

NH2

Adenosine
N Deaminase

C
N

O
C
HN

N
CH

CH
HC

HC

C
N

N
Ribose-P

Ribose-P
IMP

AMP

HC

O
C

HN
C

C
N
H

C
C

N
H
Hypoxanthine
N

HN

N
H

Uric Acid

(2,6,8-trioxypurine)

CH
O

C
N
H

N
CH

Xanthine Oxidase

HN

N
H

GMP

Xanthine

The end product of purine metabolism

Uric acid
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.
The normal concentration of uric acid in the
serum of adults is in the range of 3-7 mg/dl.

GOUT
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.

The uric acid and the gout

Out of body
In urine

Diabetese nephrosis

Hypoxanthine
Xanthine

Uric acid
Over 8mg/dl, in the plasma

Gout, Urate crystallization

in joints, soft tissue, cartilage and kidney

Advanced Gout
Clinically Apparent Tophi
2

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.

Allopurinol a suicide inhibitor used to treat Gout

O

HN

HN

H
C
N

CH
HC

N
H
Hypoxanthine
N

HC

C
N
Allopurinol

Xanthine oxidase
Xanthine oxidase

N
H

Section 4
Synthesis of Pyrimidine Nucleotides

 4.1 De novo synthesis

shorter pathway than for purines
Pyrimidine ring is made first, then attached to
ribose-P (unlike purine biosynthesis)
only 2 precursors (aspartate and glutamine, plus
HCO3-) contribute to the 6-membered ring
requires 6 steps (instead of 11 for purine)
the product is UMP (uridine monophosphate)

1. Element source of pyrimidine

base
C
Gln
CO2

N3
C2

5C
6C

Asp

Step 1: synthesis of carbamoyl

phosphate

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 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
dihydroorotate

Step 4: oxidation of
dihydroorotate to orotate

CoQ
QH2

(a
pyrimidine)

Step 5: acquisition of ribose phosphate

moiety

Step 6: decarboxylation of OMP

The big picture

3. UTP and CTP biosynthesis

kinase

UMP
ATP

UDP
ADP

ATP

kinase

UTP
ADP

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.
UDP

dUDP

dCMP

dCDP

dUMP
N5,N10-methylenetetrahydrofolic Acid
dTMP synthetase

dTMP

ATP

ATP

ADP

dTDP

dTTP

ADP

dTMP synthesis at the nucleoside

monophosphate level.
dUDP
H2O

O
Pi

NH3 O
H2O

dCMP

thymidylate synthase

HN

HN
O

N
d R 5' P

d R 5' P
dUMP

CH3

FH2
reductase
NADP+

NADPH
+ H+

dTMP

 4. 2 Salvage pathway
uridine-cytidine kinase
uridine
cytidine + ATP
deoxythymidine + ATP
deoxycytidine + ATP
uracil
thymine + PRPP
orotic acid

thymidine kinase
deoxycytidine kinase
pyrimidine phosphate
ribosyltransferase

UMP + ADP
CMP
dTMP + ADP
dCMP + ADP
UMP
dTMP + PPi
OMP

 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.
O
F

HN
O

N
H
5-FU

CH3

HN
O

N
H
thymine

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.
NH2

NH2
N

N
O
CH2OH
O
H

OH

H
H

OH

O
CH2OH
O

ara-c

H
OH

OH

cytosine

Section 5
Degradation of Pyrimidine Nucleotides

NH2
N
O

O
H2O

N
H
cytosine

NH3

HN
O

uracil

HN
N
H

HOOC

N
H

N
H

thymine

HOOC

NH2 CH2

-ureidopropionate

CH3

CH2

NH2 CH CH3
O

CH2 -ureidoN
isobutyrate
H

H2O
H2N CH2 CH2 COOH
-alanine

H2O

CO2 + NH3

H2N CH2 CH COOH

CH3
-aminoisobutyrate

Highly soluble products

Summary of purine biosynthesis

dADP

dATP

AMP

ADP

ATP

GMP

GDP

GTP

dGDP

dGTP

IMP

Summary of pyrimidine biosynthesis

dTTP

dTMP

dTDP

dUMP

dUDP

UMP

UDP

UTP

CDP

CTP

dCDP

dCTP

dCMP

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, LeschNyhan 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
pyrimidine bases
Salvage pathway
Antimetabolites of pyrimidine nucleotides

Catabolism of Pyrimidine Nucleotides