Biochemistry Ⅱ
Zhihong Li（李志红）
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
N
H
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-b-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
N10-Formyltetrahydrofolate
N10-Formyltetrahydrofolate
First, synthesis Inosine-5'-Monophosphate, IMP
FH4 (or THF)
N10—CHO—FH4
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
1
ATP
AMP
2
ribose phosphate pyrophosphokinase
Step 2: acquisition of purine atom N9
Gln:PRPP amidotransferase
•Steps 1 and 2 are tightly
regulated by feedback inhibition
5-磷酸核糖胺,PRA
Step 3: acquisition of purine atoms C4, C5, and N7
3
glycinamide synthetase
甘氨酰胺核苷酸
•Step 4: acquisition of purine atom C8
4
GAR transformylase
甲酰甘氨酰胺核苷酸
Step 5: acquisition of purine atom N3
5
甲酰甘氨咪核苷酸
•Step 6: closing of the imidazole ring
6
5-氨基咪唑核苷酸
Step 7: acquisition of C6
7
AIR carboxylase
Carboxyaminoimidazole
ribonucleotide (CAIR)
5-氨基-4-羧基咪唑核苷酸
Step 8: acquisition of N1
Carboxyaminoimidazole
ribonucleotide (CAIR)
SAICAR synthetase
5-氨基-4-(N-琥珀酸)
-甲酰胺咪唑核苷酸
Step 9: elimination of fumarate
adenylosuccinate lyase
5-氨基-4-甲酰胺咪唑核苷酸
Step 10: acquisition of C2
AICAR transformylase
5-甲酰胺基-4-甲酰胺咪唑核苷酸
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
N
Hypoxanthine
O
N
N
hypoxanthine-guanine
phosphoribosyl transferase
(HGPRT)
PRPP
N
N
Guanine
NH2
3POH2C
O
N
N
N
HO OH
IMP
O
PPi
N
2-O
3POH2C
O
N
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.
P
P
O CH2 O
Base
ribonucleotide
reductase
P
P
O CH2 O
Mg2+
Base
H2O
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.
OH
SH
N
N
N
N
H
hypoxanthine
N
N
N
N
H
6-MP
• 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.
O
H2N
NH2
C
CH2
CH2
O
N
N
CH2
C
CH COOH
Gln
NH2
O
CH2
CH COOH
AS
3. Folic acid analogs
• Aminopterin (AP) and Methotrexate (MTX)
NH2
N
N
H2N
N
CH2
R
O
N
C NH C CH2
H
OH
H2N
H
N
N
CH2 COOH
N
MTX
R＝CH3： TXT
R＝H： AP
N
COOH
CH2 N
O
COOH
C NH
C
H
CH2 CH2
N
folic acid
COOH
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
C
O
C
HN
C
N
CH
CH
HC
C
HC
N
N
C
N
O
N
Ribose-P
Ribose-P
IMP
AMP
C
HN
CH
HC
C
C
N
HN
C
C
O
C
N
H
N
H
Hypoxanthine
O
C
HN
C
N
Xanthine Oxidase
O
C
N
H
Uric Acid
(2,6,8-trioxypurine)
C
N
O
CH
C
O
C
N
H
N
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
Hypoxanthine
Xanthine
Uric acid 
Over 8mg/dl, in the plasma
Diabetese nephrosis
……
Gout, Urate crystallization
in joints, soft tissue, cartilage and kidney
Advanced Gout
Clinically Apparent Tophi
2
1
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.
3
Allopurinol – a suicide inhibitor used to treat Gout
O
O
C
C
HN
C
N
HN
C
H
C
N
CH
HC
C
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
N3
4
5C
Asp
CO 2
C2
1
N
6C
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
kinase
UMP
UDP
ATP
ADP
ATP
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
O
Pi
NH3 O
H2O
dCMP
thymidylate synthase
HN
HN
O
N
CH3
N
5
10
FH2
N
,
N
-CH2-FH4
d R 5' P
d R 5' P
FH2
dTMP
dUMP
NADPH
reductase
+ H+
FH4
NADP+
§ 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
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
N
OH
H
H
H
OH
O
CH2OH
O
H
ara-c
N
H
H
H
OH
OH
cytosine
Section 5
Degradation of Pyrimidine Nucleotides
O
NH2
N
O
H2O
N
H
cytosine
NH3
O
HN
O
uracil
HN
N
H
O
HOOC
O
N
H
N
H
thymine
HOOC
NH2 CH2
¦Â-ureidopropionate
CH3
CH2
NH2 CH CH3
O
CH2 ¦Â-ureidoN
isobutyrate
H
H2O
H2N CH2 CH2 COOH
¦Â-alanine
H2O
CO2 + NH3
H2N CH2 CH COOH
CH3
¦Â-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
dCMP
dCDP
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, 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