Seminar 11 - zakladbiochemii-2wl.wum.edu.pl · • De novo synthesis ... intermediate of purine...
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Transcript of Seminar 11 - zakladbiochemii-2wl.wum.edu.pl · • De novo synthesis ... intermediate of purine...
Nitrogenous base + ribose
Nitrogenous base + ribose + phosphate
Nucleoside =
Nucleotide =
Nucleoside and Nucleotide
Nucleoprotein
Nucleic acid Protein
Nucleotide
Nucleoside Phosphate
Base Ribose
Nucleotidase
Nucleosidase
In stomach Gastric acid and pepsin
In small intestine Endonucleases: RNase and DNase
Degradation of nucleic acid
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
Significances of 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
• Salvage pathways
The synthesis of nucleotide by recycle the free bases or
nucleosides released from nucleic acid breakdown
Synthesis of Purine Nucleotides
Site
• in cytosol of liver, small intestine and thymus
Characteristics
• Purines are synthesized using 5-phosphoribose (R-5-P)
as the starting material step by step
• PRPP (5-phosphoribosyl-1-pyrophosphate) is active
donor of R-5-P
• AMP and GMP are synthesized further at the base of
IMP (Inosine-5-Monophosphate)
De novo synthesis
• basic pathway for biosynthesis of purine
ribonucleotides
• starts from ribose-5-phosphate (R-5-P)
• Requires 11 steps overall
• occurs primarily in the liver
Synthesis of Inosine Monophosphate (IMP)
OH
1 ATP
AMP
2
Gln:PRPP amidotransferase
ribose phosphate pyrophosphokinase / PRPP synthetase
Step 1: Activation of ribose-5-phosphate
Step 2: Acquisition of purine atom N9
(PRA)
Steps 1 and 2 are tightly regulated by feedback inhibition
Step 11: Ring closure to form IMP
Once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells)
Note: GTP is used for AMP synthesis
Note: ATP is used for GMP synthesis
IMP is the precursor for both AMP and GMP
Conversion of IMP to AMP and GMP
kinase
ADP
kinase
ADP
ATP
ATP ADP
AMP
ATP
kinase
GDP
kinase
ADP
GTP
ATP ADP
GMP
ATP
ADP, ATP, GDP and GTP biosynthesis
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]
Regulation of de novo synthesis
• 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
Salvage pathway
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
• 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
Formation of deoxyribonucleotide
S
S
H2OMg
2+
NADPH + H+
NADP+
SH
SHthioredoxin
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
• 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
Antimetabolites of purine nucleotides
• 6-Mercaptopurine (6-MP) is a analog of hypoxanthine
N
N NH
N
OH
N
N NH
N
SH
6-MPhypoxanthine
Purine analogs
6-MP 6-MP nucleotide
de novo synthesis
salvage pathway
HGPRT
amidotransferase
IMP
AMP and GMP
-
-
-
-
-
6-MP nucleotide is a analog of IMP
• Azaserine (AS) is a analog of Gln (glutamine)
H2N C CH2
O
CH2 CH
NH2
COOH Gln
C
O
CH2 CH
NH2
COOH ASNN CH2 O
Amino 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
Folic acid analogs
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.
N
HC
N
C
C
C
N
CH
N
NH2
Ribose-P
AMP
HN
HC
N
C
C
C
N
CH
N
O
Ribose-P
IMPHN
HC
N
C
C
C
NH
CH
N
O
HN
C
NH
C
C
C
NH
CH
N
O
O
HN
C
NH
C
C
C
NH
C
N
O
O
O
GMP
Hypoxanthine
Uric Acid Xanthine
Xanthine Oxidase
(2,6,8-trioxypurine)
Adenosine deaminase
Xanthine oxidase
The end product of purine metabolism
• 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
Uric acid
• 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 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 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 Gout Clinically 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.
2 1
3
HN
HC
N
C
C
C
NH
CH
N
O
Hypoxanthine
HN
HC
N
C
C
C
NH
N
HC
O
Allopurinol
Allopurinol – a suicide inhibitor used to treat Gout
Xanthine oxidase
Xanthine oxidase
• 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)
De novo synthesis
• 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
• the immediate precursor of thymidylate (dTMP) is dUMP
• the formation of dUMP either by deamination of dCMP or by
hydrolyzation of dUDP
dTMP dTDP dTTP
dUMP
dUDP dCMP dCDP
N5,N10-methylene-
tetrahydrofolic Acid
ATP ATP
ADP ADP
dTMP synthetase
UDP
Formation of dTMP
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, N
10-CH2-FH4 FH2
dTMP synthesis at the nucleoside monophosphate level
carbamoyl phosphate
carbamoyl aspartate
UMP
ATP + CO2 + Gln
PRPP
UTP CTP
ATP + R-5-P
purine nucleotide
pyrimidine nucleotide
Regulation of de novo synthesis
+ 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
Salvage pathway
• antimetabolites of pyrimidine nucleotides are similar with them of purine nucleotides
Antimetabolites of pyrimidine nucleotides
• 5-fluorouracil (5-FU) is a analog of thymine
HN
NH
O
O
FHN
NH
O
O
CH3
thymine5-FU
Pyrimidine analogs
• Azaserine (AS) inhibits the synthesis of CTP
Amino acid analogs
• Methotrexate (MTX) inhibits the synthesis of dTMP
Folic acid 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
Nucleoside analogs
H2OH2O
H2N CH2 CH2 COOH H2N CH2 CH COOH
CH3
N
NH
O
NH2
H2O NH3HN
NH
O
O
CH2
CH2NH2
NH
O
HOOC
HN
NH
O
O
CH3
CH2
CHNH2
NH
O
HOOC
CH3
cytosine uracilthymine
¦Â-ureidopropionate
¦Â-ureido-isobutyrate
CO2 + NH3
¦Â-alanine ¦Â-aminoisobutyrate
Highly soluble products