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Biosynthesis of nucleotides
Phar 6152
Spring 2004
Natalia Tretyakova, Ph.D.
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.
2.
3.
4.
5.
a.
b.
c.
Building blocks of nucleic acids (DNA and RNA).
Involved in energy storage, muscle contraction,
active transport, maintenance of ion gradients.
Activated intermediates in biosynthesis
(e.g. UDP-glucose, S-adenosylmethionine).
Components of coenzymes (NAD+, NADP+, FAD,
FMN, and CoA)
Metabolic regulators:
Second messengers (cAMP, cGMP)
Phosphate donors in signal transduction (ATP)
Regulation of some enzymes via adenylation and
uridylylation
Nucleotides
Nucleotide
Purine or
Pyrimidine
Base
O
5'
-O
P
b-glycosidic bond
O
O
O-
H
4'
3'
H
Phosphate
H
O
1'
2' H
H(OH)
Pentose sugar
Nucleoside
RNA- ribose (R)
DNA – deoxyribose (dR)
Nucleobase structures
O
O
NH2
6
7
N
5
9N
4
N
1
N
N
N
N
O
N
NH
NH
NH
8
2
N
H
N
H
N
H
N
N
H
N
3
Purine
Adenine (A)
Hypoxanthine
NH2
O
NH2
N
Guanine (G)
3
O
6
H3C
N
N
NH
NH
2
N1
H
Pyrimidine
N
H
O
Cytosine (C)
N
H
Thymine (T)
O
N
H
Uracil (U)
N
H
Xanthine
4
5
N
H
O
O
Hypoxanthine
Xanthine
Inosine
Xanthosine
Inosinate (IMP)
Xanthylate (XMP)
Two major routes for nucleotide
biosynthesis
dNTPs
Stryer Fig. 25.1
dNTPs
Nucleobase Products of Intracellular or
dietary/intestinal degradation can be recycled via
salvage pathways 1 and 2 (red)
1
2
Phosphoribosyl transferases involved in salvage
pathway convert free bases to nucleotides
adenine phosphoribosyl
transferase
Adenylate + PPi
Adenine + PRPP
hypoxanthine-guanine
phosphoribosyl transferase
Guanylate + PPi
Guanine + PRPP
Inosinate + PPi
Hypoxanthine + PRPP
2-
2-
O3PO
CH2
O
-O
Base +
O
-O
P
O
OH
(HGPRT)
O3PO
CH2
O-
Base
O
+ PPi
P
O
O
OH
5-phosphoribosyl-1-pyrophosphate (PRPP)
OH
OH
Biodegradation of Nucleotides
(Stryer p. 709-711)
Degradation
Salvage
2-
O3PO
HO
5'
Base
O
H
H
OH
H
OH
H
Nucleotide 5'-phosphate
Nucleootidase
O
Base
nucleoside phosphorylase
H
H
OH
H
OH
H
Base
HO
O
H
H
Nucleoside
OPO32-
H
OH
OH
Ribose-1-phosphate
phosphoribo
mutase
2-
O3PO
OH
O
PRPP
H
H
OH
OH
H
Ribose-5-phosphate
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
O
N
N
H
NH
N
Hypoxanthine
O
O
NH
N
N
H
N
Allopurinol
NH
N
N
H
N
H
O
Alloxanthine
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
Lack of HGPRT activity in Lesch-Nyhan
Syndrome causes a buildup of PRPP, which
activates the synthesis of purine nucleotides
hypoxanthine-guanine
phosphoribosyl transferase
Guanine + PRPP
Hypoxanthine + PRPP
Guanylate + PPi
Inosinate + PPi
•Excessive uric acid forms as a degradation
product of purine nucleotides
•Basis of neurological aberrations is unknown
Immunodeficiency induced by
Adenosine Deaminase defects
AMP
deaminase
• 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)
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 clinical
disease.
Key concepts in Biosynthesis: Review
•Committed step
•Regulated step
•Allosteric inhibitor
•Feedback inhibition
De novo Biosynthesis of
Pyrimidines
NH2
O
N
N
O
-O
P
NH
O
N
O
O
-O
O
H
O-
P
O
OH
OH
O
-O
O
H
OH
NH
N
O
H
H
O
H
H
OH
O
O
OH
OH
P
O
H
H
OH
H
H
H
Required reading: Stryer’s Biochemistry 5th Ed., p. 262-267, 694-698
De novo Biosynthesis of Pyrimidines
O
NH2
O
CH3
HN
N
HN
O
N
H
Thymine (T)
O
N
H
Cytosine (C)
O
N
H
Uracil (U)
O
NH2
C
O
-O
C
CH2
O
H2N
PO3-
2-
O3PO
CH
COO-
CH2
O
-O
O
-O
P
O
OH
Stryer Fig. 25.2
dTTP
OH
O-
P
O
O
Part 1. The formation of carbamoyl phosphate
O-O
P
O
O
C
O
NH2
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:
CPS
General strategy for making C-N bonds: C-OH is
phosphorylated to generate a good leaving group (phosphate)
General Mechanism for making C-N bonds:
R
R
R'
O-
ADP R
OH
O
R'
ATP
O
R'
P
O
ONH3
O
R'
Pi R
O-
R
NH2
P
O-
NH2
O
R'
Ammonia necessary for the formation of carbamic
acid originates from glutamine:
NH2
H2
C
H2N
C
O
CH
C
H2
-
C
O
O
Glutamine (Gln)
CPS
H2
C
-
NH3
NH2
O
C
O
CH
C
H2
Glutamate
C
O
O-
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
H2
C
H2N
C
O
CH
C
H2
C
O-
O
Glutamine (Gln)
Stryer Fig. 25.4
CPS
NH3
NH2
H2
C
-
O
C
O
CH
C
H2
Glutamate
C
O
O-
Carbamic acid is phosphorylated
CPS
Substrate channeling in CPS
NH2
H2
C
H2N
C
O
CH
C
H2
-
C
O
O
Glutamine (Gln)
Stryer Fig. 25.5
CPS
NH3
NH2
H2
C
-
O
C
O
CH
C
H2
Glutamate
C
O
O-
Carbamoyl phosphate supplies the C-2 and the N-3 of the pyrimidine ring
O
NH2
C
O
-O
O
C
CH2
H2N
PO3-
dTTP
CH
COO-
Part 2. The formation of orotate.
Aspartate is coupled to carbamoyl phosphate
O
Asp replaces Pi
Pi
O-O
P
HN
O
O
C
O
OOC
NH2
Carbamoyl phosphate
CH
Asp
CH
NH2
COO
C
H2
Carbamoylaspartate
NH2
-OOC
C
COOC
H2
Enzyme: aspartate transcarbamoylase
This is the committed step in pyrimidine biosynthesis
Aspartate transcarbamoylase is allosterically inhibited by CTP
Stryer Fig. 10.2
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
O
O
HN
OOC
CH
C
+
H
H2O
NH2
COO
C
Dihydroorotase
H2
N-Carbamoylaspartate
HN
OOC
C
CH
HN
OOC
CH
N
H
H
O
C
O-
C
H2
Tetrahedral transition state
C
C
H2
Dihydroorotate
O
C
NH
- H2O
O
Dihydroorotate is oxidized to orotate
Dihydroorotate dehydrogenase
Part 3. The formation of UMP
a. Orotate is phosphoribosylated to OMP
O
Pyrimidine phosphoribosyl
transferase
O
-
C
HN
OOC
NH
C
O3PO
CH2
O
-O
O
-O
P
C
C
H
-
O
O
OH
Orotate
O-
C
COO
OH
OH
Orotidylate
(orotidine monophosphate, OMP)
PRPP synthetase
ATP
O3PO
OH
N
O
OH
AMP
CH2
CH
C
O
5-phosphoribosyl-1-pyrophosphate (PRPP)
2-
O
CH2
P
O
PPi O3PO
C
HN
O
OH
OH
ribose-5-phosphate
(from pentose phosphate pathway)
b. OMP is decarboxylated to form UMP
OMP decarboxylase
(UMP synthetase)
(OMP)
(UMP)
Note: phosphoribosyl transfer and decarboxylase
activities are co-localized in UMP synthetase
c.Phosphorylation of UMP gives rise to UDP and
UTP:
UMP kinase
UMP + ATP
UDP + ADP
nucleotide diphosphate kinase
UDP + ATP
UTP + ADP
CTP is produced by replacing the 4-keto group of UTP
with NH2
CTP synthetase
O
O
C4
N
4-
O3PO3PO3PO-H2C
O
CH2
OH
O-
OCH
C
N
O
P
C
H
OH
Note: TTP for DNA synthesis is produced via methylation of CTP (will
discuss later)
Regulation of pyrimidine nucleotide
biosynthesis
Glutamine + HCO3- + ATP
Regulated step
PRPP
Carbamoyl phosphate
Committed step
Carbamoyl aspartate
OMP
Carbamoyl phosphate
synthetase
Aspartate
transcarbamoylase
OMP decarboxylase
(UMP synthetase)
UMP
UDP
UTP
CTP
CTP synthetase
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
O
C
HN
OOC C
-
O3PO
NH
CH2
O
-O
-O
P
C
C
H
Orotate
O
O
O
OH
O-
PPi
P
O
O
OH
5-phosphoribosyl-1-pyrophosphate (PRPP)
– Treatment: patients are fed uridine
U  UMP  UDP  UTP
UTP inhibits carbamoyl phosphate synthase II, preventing
the biosynthesis and accumulation of orotic acid
UTP inhibits carbamoyl phosphate synthase II, preventing the
biosynthesis and accumulation of orotic acid
Glutamine + HCO3- + ATP
PRPP
Carbamoyl phosphate
Carbamoyl aspartate
OMP
UMP
UDP
UTP
CTP
Carbamoyl phosphate
synthetase
Drug inhibitors of pyrimidine biosynthesis
Inhibitors of PRPP synthetase:
2-
AMP
ATP
O3PO
CH2
-
O3PO
O
CH2
O
-O
O
OH
OH
OH
OH
ribose-5-phosphate
(from pentose phosphate pathway)
NH2
OCH3
N
N
N
N
N
N
N
N
HO
NH
O
H
H
HO
NH
O
H
H
OH
OH
MRPP (MP)
noncompetitive, Ki = 190 M
-O
P
PRPP synthetase
OH
O
H
H
H
OH
H
OH
ARPP (MP)
noncompetitive, Ki = 430 M
O-
P
O
O
Inhibitors of dihydroorotase
O
O
C
C
HN
OOC
CH
NH2
HN
COO
OOC
C
H2
O
H+
H
H2O
N H
CH
C
H2
HN
NH
OOC CH
OO-
C
C
Carbamoylaspartate
C
C
H2
Dihydroorotate
Tetrahedral Transition State
-O
O
HS
O
SH
P
HC
OOC CH
O
NH
HC
C
N
H
HDDP
Ki = 0.74 M
O
OOC CH
HC
NH
OOC CH
C
N
H
O
MMDHO
Ki = 0.14 M
CH
C
N
H
O
MOAC
Ki = 2.9 M
Pyrimidine biosynthesis: take home message
1.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.