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Biosynthesis of nucleotides
Natalia Tretyakova, Ph.D.
Phar 6152
Spring 2004
Required reading: Stryer’s Biochemistry 5th edition, p. 262-268, 693-712
(or Stryer’s Biochemistry 4th edition p. 238-244, 739-759)
Tentative Lecture plan: Biosynthesis of Nucleotides
03-31 Introduction. Biological functions and sources of nucleotides. Nucleotide metabolism.
04-02 Biosynthesis of pyrimidine ribonucleotides.
04-05 Biosynthesis of purine ribonucleotides
04-07 Biosynthesis of deoxyribonucleotides. Inhibitors of nucleotide metabolism as drugs.
04-09 Review
04-12 Exam
Biological functions and sources of nucleotides.
Nucleotide metabolism
Required reading: Stryer’s Biochemistry 5th Ed., p. 693-694, 709-711
Biological functions of nucleotides
1. Building blocks of nucleic acids (DNA and RNA).
2. Involved in energy storage, muscle contraction, active transport, maintenance of ion gradients.
3. Activated intermediates in biosynthesis (e.g. UDP-glucose, S-adenosylmethionine).
4. Components of coenzymes (NAD+, NADP+, FAD, FMN, and CoA)
5. Metabolic regulators:a. Second messengers (cAMP, cGMP)b. Phosphate donors in signal transduction (ATP)
c. Regulation of some enzymes via adenylation and uridylylation
-OO
H(OH)
HH
HHO
OP
O
O-
Purine orPyrimidineBase
Phosphate
Pentose sugar
Nucleoside
Nucleotide
1'
2'3'
4'
5'
Nucleotides
-glycosidic bond
RNA- ribose (R)DNA – deoxyribose (dR)
NN
NNH
NH2
NNH
NH
NH
O
O
N
NH
NH2
O
H3C
NH
NH
O
O
XanthineAdenine (A)
Thymine (T)Cytosine (C)
NH
NH
O
O
Uracil (U)
NN
NNH
Purine
N
NH
Pyrimidine
1
2
3
4
5
6
3
2
16
4
57
8
9
NNH
NNH
O
NH2
Guanine (G)
NNH
NNH
O
Hypoxanthine
Nucleobase structures
Hypoxanthine Inosine Inosinate (IMP)Xanthine Xanthosine Xanthylate (XMP)
Two major routes for nucleotide biosynthesis
dNTPs
dNTPs
Stryer Fig. 25.1
Nucleobase Products of Intracellular or dietary/intestinal degradation can be recycled via
salvage pathways 1 and 2 (red)
1 2
Adenine + PRPP Adenylate + PPi
adenine phosphoribosyl transferase
Guanine + PRPP Guanylate + PPi
hypoxanthine-guanine phosphoribosyl transferase
Hypoxanthine + PRPP + PPi
O
OHOH
CH2
OP
OP
O-O
-OO-
2- O3PO
O
5-phosphoribosyl-1-pyrophosphate (PRPP)
Base +
Phosphoribosyl transferases involved in salvage pathway convert free bases to nucleotides
(HGPRT)
Inosinate
O
OHOH
CH2
2- O3PO Base
+ PPi
Biodegradation of Nucleotides
Base
2-O3PO
O
OHOH
HH
HH
NucleootidaseBase
HO
O
OHOH
HH
HH
Base
HO
O
OHOH
HH
H OPO32-
Salvage Degradation
2-O3PO
O
OHOH
HH
H
Nucleotide 5'-phosphate Nucleoside
Ribose-1-phosphate
Ribose-5-phosphate
nucleoside phosphorylase
phosphoribomutase
PRPP
OH
5'
(Stryer p. 709-711)
Nucleobase Products of Intracellular or dietary/intestinal degradation can be recycled via
salvage pathways 1 and 2 (red)
1 2
Purine biodegradation in humans leads to uric acid
AMP is deaminated to IMP
AMP deaminase
IMP is deribosylated to hypoxanthine
phosphorylase
Hypoxanthine is oxidized to xanthine
Guanine can be deaminated to give xanthine
Uric acid is the final product of purine degradation in mammals
Uric acid is excreted as urate
Deleterious consequences of defective purine metabolism
• Gout (excess accumulation of uric acid)
• Lesch-Nyhan syndrome (HGPRT null)
• Immunodeficiency
Gout
• Precipitation and deposition of uric acid causes arthritic pain and kidney stones
• Causes: impaired excretion of uric acid and deficiencies in HGPRT
NH
NNH
N
O
Hypoxanthine
NH
NNH
N
O
Allopurinol
NH
NH
NH
N
O
Alloxanthine
O
Lesch-Nyhan Syndrome
• Caused by a severe deficiency in HGPRT activity
• Symptoms are gouty arthritis due to uric acid accumulation and severe neurological malfunctions including mental retardation, aggressiveness, and self-mutilation
• Sex-linked trait occurring mostly in males
Guanine + PRPP Guanylate + PPi
hypoxanthine-guanine phosphoribosyl transferase
Hypoxanthine + PRPP Inosinate + PPi
Lack of HGPRT activity in Lesch-Nyhan Syndrome causes a buildup of PRPP, which activates the synthesis of purine nucleotides
•Excessive uric acid forms as a degradation product of purine nucleotides
•Basis of neurological aberrations is unknown
Immunodeficiency induced by Adenosine Deaminase defects
• Defects in AMP deaminase prevent biodegradation of AMP• AMP is converted into dATP• dATP inhibits the synthesis of deoxyribonucleotides by
ribonucleotide reductase, causing problems with the immune
system (death of lymphocytes, immunodeficiency disease)
AMPdeaminase
Summary:
• Nucleotides have many important functions in a cell.
• Two major sources of nucleotides are salvage pathway and de novo biosynthesis
•Purine nucleotides are biodegraded by nucleotidases,
nucleotide phosphorylases, deaminases, and xanthine oxidase.
•Uric acid is the final product of purine biodegradation in mammals
• Defective purine metabolism leads to clinicaldisease.
Key concepts in Biosynthesis: Review
•Committed step
•Regulated step
•Allosteric inhibitor
•Feedback inhibition
De novo Biosynthesis of Pyrimidines
Required reading: Stryer’s Biochemistry 5th Ed., p. 262-267, 694-698
-O
N
NH2
ON
O
OHOH
HH
HH
OP
O-
O
-O
NH
O
ON
O
OHOH
HH
HH
OP
O-
O
-O
NH
O
ON
O
HOH
HH
HH
OP
O-
O
De novo Biosynthesis of Pyrimidines
NH2
CO O
PO3-H2N
CH
COO-
CH2
C-O
O
N
NH
NH2
O
CH3HN
NH
O
O
Thymine (T) Cytosine (C)
HN
NH
O
O
Uracil (U)
O
OHOH
CH2
OP
OP
O-O -O O-
2- O3PO
O
dTTPStryer Fig. 25.2
Part 1. The formation of carbamoyl phosphate
OC
NH2
O
PO
-O
O-
Carbamoyl phosphate
Enzyme: carbamoyl phosphate synthetase II (CPS)This is the regulated step in pyrimidine biosynthesis
Bicarbonate is phosphorylated
CPS
Phosphate is displaced by ammonia:
:
General strategy for making C-N bonds: C-OH isphosphorylated to generate a good leaving group (phosphate)
CPS
General Mechanism for making C-N bonds:
O
R
R'
OH
R
R'
ATP ADP
O
R
R'
P
O-
O-
O
NH3
O
R
R'
P
O-
O-
O
NH2
Pi
NH2
R
R'
NH2
CHCC
H2
O-
O
H2C
C
H2N
O
CPSNH3
Glutamine (Gln) Glutamate
NH2
CHCC
H2
O-
O
H2C
C
-O
O
Ammonia necessary for the formation of carbamic acid originates from glutamine:
Structure of Carbamoyl phosphate synthetase II
Stryer Fig. 25.3
The active site for glutamine hydrolysis to ammonia
contains a catalytic dyad of Cys and His residues
NH2
CHCC
H2
O-
O
H2C
C
H2N
O
CPSNH3
Glutamine (Gln) Glutamate
NH2
CHCC
H2
O-
O
H2C
C
-O
O
Stryer Fig. 25.4
Carbamic acid is phosphorylated
CPS
Substrate channeling in CPS
NH2
CHCC
H2
O-
O
H2C
C
H2N
O
CPSNH3
Glutamine (Gln) Glutamate
NH2
CHCC
H2
O-
O
H2C
C
-O
O
Stryer Fig. 25.5
Carbamoyl phosphate supplies the C-2 and the N-3 of the pyrimidine ring
NH2
CO O
PO3-H2N
CH
COO-
CH2
C-O
O
dTTP
Part 2. The formation of orotate.
Aspartate is coupled to carbamoyl phosphate
Enzyme: aspartate transcarbamoylase
OC
NH2
O
PO
-OO-
Pi
NH2
CH-OOC
CH2
COO-
CNH2
O
HN
CHOOC
CH2
COO
Carbamoyl phosphateCarbamoylaspartate
Asp replaces Pi
Asp
This is the committed step in pyrimidine biosynthesis
Stryer Fig. 10.2
Aspartate transcarbamoylase is allosterically inhibited by CTP
Allosteric regulation of Aspartate Transcarbamoylase
Stryer Fig. 10.5
PALA is a bisubstrate analog that mimics the reaction intermediate on the way to carbamoyl aspartate
Bisubstrate analog
PALA binds to the active site within catalytic subunit
Stryer Fig. 10.7
Substrate binding to Aspartate Transcabamoylase induces a large change in ATC quaternary structure
Stryer Fig. 10.8
CTP binding prevents ATC transition to the active R state
Stryer Fig. 10.9
Allosteric regulation of Aspartate Transcabamoylase
Stryer Fig. 10.10
N-Carbamoylaspartate cyclizes to dihydroorotate
CN
O
HN
CHOOCCH2
CO-
Tetrahedral transition state
O-
H
H
CNH2
O
HN
CHOOC
CH2
COO
H+H2O C
NH
O
HN
CHOOCCH2
CODihydroorotase
N-Carbamoylaspartate Dihydroorotate
- H2O
Dihydroorotate dehydrogenase
Dihydroorotate is oxidized to orotate
C
HN
O
N C
COO
CH
C
O
O
OHOH
CH2
-O3POO
OHOH
CH2
OP
OP
O-O
-OO-
-O3PO
O
CNH
O
HN
COOCCH
CO
PPi
Orotate
Orotidylate(orotidine monophosphate, OMP)
5-phosphoribosyl-1-pyrophosphate (PRPP)
Part 3. The formation of UMP
a. Orotate is phosphoribosylated to OMP
Pyrimidine phosphoribosyltransferase
O
OHOH
CH2
OH
2- O3POATP
AMP
ribose-5-phosphate(from pentose phosphate pathway)
PRPP synthetase
b. OMP is decarboxylated to form UMP
(OMP) (UMP)
OMP decarboxylase(UMP synthetase)
Note: phosphoribosyl transfer and decarboxylase activities are co-localized in UMP synthetase
c.Phosphorylation of UMP gives rise to UDP and UTP:
UMP + ATP UDP + ADP
UMP kinase
UDP + ATP UTP + ADP
nucleotide diphosphate kinase
CTP is produced by replacing the 4-keto group of UTP with NH2
Note: TTP for DNA synthesis is produced via methylation of CTP (will discuss later)
CTP synthetase
C
N
O
N C
H
CH
C
O
O
OHOH
CH2
4- O3PO3PO3PO-H2C
P O-
O-
O
4
Regulation of pyrimidine nucleotide biosynthesis
PRPP
OMP
UMP
UDP
UTP
CTP
Glutamine + HCO3- + ATP
Carbamoyl phosphate
Carbamoyl aspartate
OMP decarboxylase(UMP synthetase)
Carbamoyl phosphate synthetase
CTP synthetase
Aspartate transcarbamoylase
Regulated step
Committed step
Defects in de novo pyrimidine biosynthesis lead to clinical disease
• Orotic acidurea– Symptoms: anemia, growth retardation, orotic acid excretion– Causes: a defect in phosphoribosyl transferase or orotidine
decarboxylase
– Treatment: patients are fed uridineU UMP UDP UTP
UTP inhibits carbamoyl phosphate synthase II, preventing the biosynthesis and accumulation of orotic acid
O
OHOH
CH2
OP
OP
O-O -O O-
-O3PO
O
CNH
O
HN
COOCCH
CO
PPi
Orotate 5-phosphoribosyl-1-pyrophosphate (PRPP)
UTP inhibits carbamoyl phosphate synthase II, preventing the biosynthesis and accumulation of orotic acid
PRPP
OMP
UMP
UDP
UTP
CTP
Glutamine + HCO3- + ATP
Carbamoyl phosphate
Carbamoyl aspartate
Carbamoyl phosphate synthetase
Drug inhibitors of pyrimidine biosynthesis
Inhibitors of PRPP synthetase:
N
NN
N
NHO
OHOH
HH
HH
HO
OCH3
N
NN
N
NHO
OHOH
HH
HH
HO
NH2
MRPP (MP)noncompetitive, Ki = 190 M
ARPP (MP)noncompetitive, Ki = 430 M
O
OHOH
CH2
OH
2- O3PO ATP AMP
ribose-5-phosphate(from pentose phosphate pathway)
PRPP synthetase
O
OHOH
CH2
OP
OP
O-O
-OO-
-O3PO
O
CNH2
O
HN
CHOOC
CH2
COO
CN
O
HN
CHOOCCH2
CO-
H
H
O-
H+ H2O CNH
O
HN
CHOOCCH2
CO
Carbamoylaspartate DihydroorotateTetrahedral Transition State
Inhibitors of dihydroorotase
NHHC
CHOOCNH
CO
HS
PCH
O
HC
CHOOCNH
CO
SH
MOAC
NHHC
CHOOCNH
CO
O-O
HDDP MMDHO
Ki = 0.14 M Ki = 0.74 M Ki = 2.9 M
Pyrimidine biosynthesis: take home message1.Pyrimidines are synthesized by de novo and salvage pathways.
2. The pyrimidine ring is synthesized from pre-assembled ingredients (carbamoyl phosphate and aspartate) and then attached to the ribose.
3. Pyrimidine biosynthesis is tightly regulated via feedback inhibition (CTP synthetase, carbamoyl phosphate synthetase, aspartate transcarbamoylase) and transcriptional regulation (ATCase).
4. The mammalian enzymes are multifunctional (e.g. carbamoyl phosphate synthetase, UMP synthetase) and form multienzyme complexes to increase efficiency.
5. Drug inhibitors of pyrimidine biosynthesis are under development as potential antimicrobial and anticancer agents.
