Chapter 26
The Synthesis and Degradation of
Nucleotides
Biochemistry
by
Reginald Garrett and Charles Grisham
Outline
1.
2.
3.
4.
5.
6.
7.
Can Cells Synthesize Nucleotides?
How Do Cells Synthesize Purines?
Can Cells Salvage Purines?
How Are Purines Degraded?
How Do Cells Synthesize Pyrimidines?
How Are Pyrimidines Degraded?
How Do Cells Form the
Deoxyribonucleotides That Are Necessary for
DNA Synthesis?
8. How Are Thymine Nucleotides Synthesized?
26.1 – Can Cells Synthesize
Nucleotides?
• Nearly all organisms synthesize purines and
pyrimidines "de novo biosynthesis pathway"
• Many organisms also "salvage" purines and
pyrimidines from diet and degradative
pathways
• Ribose generates energy, but purine and
pyrimidine rings do not
• Nucleotide synthesis pathways are good
targets for anti-cancer/antibacterial
strategies
26.2 – How Do Cells Synthesize Purines?
•
•
•
•
•
John Buchanan (1948) "traced" the sources of all
nine atoms of purine ring
N-1: aspartic acid
N-3, N-9: glutamine
C-2, C-8: N10-formyl-THF - one carbon units
C-4, C-5, N-7: glycine
C-6: CO2
Figure 26.1
Nitrogen waste is excreted by birds principally as
the purine analog, uric acid.
IMP Biosynthesis
The first purine product of this pathway, IMP
(inosinic acid or inosine monophosphate)
• First step: Ribose-5-phosphate pyrophosphokinase
– PRPP synthesis from ribose-5-phosphate and ATP
– PRPP is limiting substance for purine synthesis
– But PRPP is a branch point so next step is the
committed step (fig 26.6)
• Second step: Gln PRPP amidotransferase
– Form phosphoribosyl-b-amine; Changes C-1
configuration (a→b)
– GMP and AMP inhibit this step - but at distinct sites
– Azaserine - Gln analog - inhibitor/anti-tumor
Figure 26.3
The de novo pathway for purine synthesis.
Step 1: Ribose-5-phosphate
pyrophosphokinase.
Step 2: Glutamine phosphoribosyl
pyrophosphate amidotransferase.
Step 3: Glycinamide ribonucleotide (GAR)
synthetase.
Step 4: GAR transformylase.
Step 5: FGAM synthetase (FGAR
amidotransferase).
Step 6: FGAM cyclase (AIR synthetase).
Step 7: AIR carboxylase.
Step 8: SAICAR synthetase.
Step 9: adenylosuccinase.
Step 10: AICAR transformylase.
Step 11: IMP synthase.
Figure 26.4
The structure of azaserine. Azaserine acts as an irreversible inhibitor of glutaminedependent enzymes by covalently attaching to nucleophilic groups in the glutaminebinding site.
•
Step 3: Glycinamide ribonucleotide (GAR)
synthetase
–
Glycine carboxyl condenses with amine in two
steps
1. Glycine carboxyl activated by -P from ATP
2. Amine attacks glycine carboxyl
–
•
Synthesize glycinamide ribonucleotide
Step 4: Glycinamide ribonucleotide (GAR)
transformylase
–
–
Formyl group of N10-formyl-THF is transferred
to free amino group of GAR
Yield N-Formylglycinamide ribonucleotide
• Step 5: Formylglycinamide ribonucleotide
(FGAR) amidotransferase
– Formylglycinamidine ribonucleotide (FGAM)
– FGAM synthetase
– C-4 carbonyl forms a P-ester from ATP and
active NH3 attacks C-4 to form imine
– Irreversibly inactivated by azaserine
Closure of the first ring,
carboxylation and attack by aspartate
• Step 6: FGAM cyclase (AIR synthetase)
– Produce aminoimidazole nucleotide (AIR)
– Similar in some ways to step 5. ATP activates
the formyl group by phosphorylation,
facilitating attack by N.
– In avian liver, the enzymes for step 3, 4, and 6
(GAR synthetase, GAR transformylase, and
AIR synthetase) reside on a polypeptide
• Step 7: AIR carboxylase
– The product is carboxyaminoimidazole
ribonucleotide (CAIR)
– Carbon dioxide is added in ATP-dependent
reaction
• Step 8: SAICAR synthetase
– N-succinylo-5-aminoimidazole-4-carboxamide
ribonucleotide
– Attack by the amino group of aspartate links
this amino acid with the carboxyl group
– The enzymes for steps 7 and 8 reside on a
bifunctional polypeptide in avian
• Step 9: adenylosuccinase
– The product is 5-aminoimidazole-4-carboxamide
ribonucleotide (AICAR); remove fumarate
– AICAR is also an intermediate in the histidine
biosynthetic pathway
• Step 10: AICAR transformylase
– N-formylaminoimidazole-4-carboxamide
ribonucleotide (FAICAR)
– Another 1-C addition (N10-formyl-THF)
• Step 11: IMP synthase (IMP cyclohydrolase)
– Amino group attacks formyl group to close the
second ring
– The enzymes for steps 10 and 11 reside on a
bifunctional polypeptide in avian
• 6 ATPs, but that this is really 7 ATP equivalents
• The dependence of purine biosynthesis on THF in
two steps means that sulfonamides block purine
synthesis in bacteria
Sulfa drugs, or sulfonamides, owe their antibiotic properties to their similarity to paminobenzoate (PABA),an important precursor in folic acid synthesis. Sulfonamides block
folic acid formation by competing with PABA.
AMP and GMP are synthesized from
IMP
•
IMP is the precursor to both AMP and GMP
AMP synthesis
1. Step 1: adenylosuccinate synthetase
– the 6-O of inosine is displaced by aspartate to yield
adenylosuccinate
– GTP is the energy input for AMP synthesis, whereas
ATP is energy input for GMP
2. Step 2: adenylosuccinase (adenylosuccinate lyase)
• carries out the nonhydrolytic removal of fumarate from
adenylosuccinate, leaving AMP.
• the same enzyme catalyzing Step 9 in the purine
pathway
•
GTP synthesis
1.
–
–
–
2.
–
–
•
Step 1: IMP dehydrogenase
Oxidation at C-2
NAD+-dependent oxidation
xanthosine monophosphate (XMP)
Step 2: GMP synthetase
Replacement of the O by N (from Gln)
ATP-dependent reaction; PPi
Starting from ribose-5-phosphate
–
–
8 ATP equivalents are consumed in the AMP
synthesis
9 ATP equivalents in GMP synthesis
Figure 26.5 The synthesis
of AMP and GMP from
IMP.
The regulation of purine synthesis
Reciprocal control occurs in two ways
IMP synthesis:
–
Allosterically regulated at the first two steps
1. R-5-P pyrophosphokinase:
•
ADP & GDP
2. phosphoribosyl pyrophosphate amidotransferase
•
A “series”: AMP, ADP, and ATP
•
G “series”: GMP, GDP, and GTP
•
PRPP is “feed-forward” activator
AMP synthesis:
adenylosuccinate synthetase is feedback-inhibited by
AMP
GMP synthesis:
IMP dehydrogenase is feedback-inhibited by GMP
Nucleoside diphosphate and
triphosphate
Nucleoside diphosphate: ATP-dependent kinase
–
Adenylate kinase: AMP +ATP → ADP +ADP
–
Guanylate kinase: GMP +ATP → GDP +ADP
Nucleotide triphosphate: non-specific enzyme
–
Nucleoside diphosphate kinase
GDP +ATP  GTP +ADP
NDP +ATP  NTP +ADP (N=G, C, U, and T)
26.3 – Can Cells Salvage Purines?
• Salvage pathways
– Recover them in useful form
– Collect hypoxanthine and guanine and recombine
them with PRPP to form nucleotides in the
HGPRT reaction
– Absence of HGPRT is cause of Lesch-Nyhan
syndrome (sex-linked); In Lesch-Nyhan, purine
synthesis is increased 200-fold and uric acid is
elevated in blood
• HGPRT & APRT
Hyperxanthine-Guanine PhosphoRibosylTransferase
Figure 26.7
Purine salvage by the HGPRT reaction.
26.4 – How Are Purines Degraded?
Purine catabolism leads to uric acid
• Nucleotidases and nucleosidases release ribose and
phosphates and leave free bases
– Nucleotidase: NMP + H2O → nucleoside + Pi
– Nucleosidase: nucleoside + H2O → base + ribose
– PNP: nucleoside + Pi → base + ribose-P
• The PNP products are converted to xanthine by
xanthine oxidase and guanine deaminase
• Xanthine oxidase converts xanthine to uric acid
– Note that xanthine oxidase can oxidize two different sites
on the purine ring system
• Neither adenosine nor deoxyadenosine is a substrate
for PNP
– Converted to inosine by adenosine deaminase (ADA)
Figure 26.8 The major
pathways for purine
catabolism in animals.
Catabolism of the
different purine
nucleotides converges
in the formation of uric
acid.
Severe combined immunodeficiency syndrome (SCID)
The effect of elevated levels of deoxyadenosine on purine metabolism. If ADA is
deficient or absent, deoxyadenosine is not converted into deoxyinosine as normal
(see Figure 26.8). Instead, it is salvaged by a nucleoside kinase, which converts it
to dAMP, leading to accumulation of dATP and inhibition of deoxynucleotide
synthesis (see Figure 26.24). Thus, DNA replication is stalled.
Purines nucleotide cycle
• Serve as an anaplerotic pathway in skeletal
muscle
– AMP deaminase
– Adenylosuccinate synthetase
– Adenylosuccinate lyase
Figure 26.9 The purine
nucleoside cycle for
anaplerotic replenishment of
citric acid cycle
intermediates in skeletal
muscle.
Xanthine Oxidase and Gout
• Xanthine Oxidase in liver, intestines mucosa, and
milk can oxidize hypoxanthine to xanthine and
xanthine to uric acid
• Humans and other primates excrete uric acid in the
urine, but most N goes out as urea
• Birds, reptiles and insects excrete uric acid and for
them it is the major nitrogen excretory compound
• Gout occurs from accumulation of uric acid crystals
in the extremities
• Allopurinol, which inhibits XO, is a treatment
Figure 26.10 Xanthine oxidase
catalyzes a hydroxylase-type reaction.
Figure 26.11 Allopurinol, an
analog of hypoxanthine, is a
potent inhibitor of xanthine
oxidase.
Animals other than humans
oxidize uric acid to form
excretory products
• Urate oxidase: Allantoin
• Allantoinase: Allantoic acid
• Allantoicase: Urea
• Urease: Ammonia
Figure 26.12
The catabolism of uric acid to allantoin,
allantoic acid, urea, or ammonia in
various animals.
26.5 – How Do Cells Synthesize
Pyrimidines?
• In contrast to purines, pyrimidines are not
synthesized as nucleotides
– The pyrimidine ring is completed before a ribose5-P is added
• Carbamoyl-P and aspartate are the precursors
of the six atoms of the pyrimidine ring
Figure 26.15
The de novo pyrimidine biosynthetic pathway.
de novo Pyrimidine Synthesis
• Step 1: Carbamoyl Phosphate synthesis
– Carbamoyl phosphate for pyrimidine synthesis is
made by carbamoyl phosphate synthetase II (CPS II)
– This is a cytosolic enzyme (whereas CPS I is
mitochondrial and used for the urea cycle)
– Substrates are HCO3-, glutamine (not NH4+), 2 ATP
– In mammals, CPS-II can be viewed as the committed
step in pyrimidine synthesis
– Bacteria have but one CPS; thus, the committed step
is the next reaction, which is mediated by aspartate
transcarbamoylase (ATCase)
(also called carbonyl-phosphate)
Figure 26.14
The reaction catalyzed by
carbamoyl phosphate
synthetase II (CPS II).
• Step 2: Aspartate transcarbamoylase
(ATCase)
– catalyzes the condensation of carbamoyl
phosphate with aspartate to form carbamoylaspartate
– carbamoyl phosphate represents an ‘activated’
carbamoyl group
• Step 3: dihydroorotase
– ring closure and dehydration via intramolecular
condensation
– Produce dihydroorotate
• Step 4: dihydroorotate dehydrogenase
– Synthesis of a true pyrimidine (orotate)
• Step 5: orotate phosphoribosyltransferase
– Orotate is joined with a ribose-P to form
orotidine-5’-phosphate (OMP)
– The ribose-P donor is PRPP
• Step 6: OMP decarboxylase
– OMP decarboxylase makes UMP (uridine-5’monophposphate, uridylic acid)
Metabolic channeling
• In bacteria, the six enzymes are distinct
• Eukaryotic pyrimidine synthesis involves
channeling and multifunctional polypeptides
– CPS-II, ATCase, and dihydroorotase are on a
cytosolic polypeptide
– Orotate PRT and OMP decarboxylase on the
other cytosolic polypeptide (UMP synthase)
• The metabolic channeling is more efficient
•
UTP and CTP
• Nucleoside monophosphate kinase
UMP + ATP → UDP + ADP
• Nucleoside diphosphate kinase
UDP + ATP → UTP + ADP
• CTP sythetase forms CTP from UTP and ATP
Regulation of pyrimidine biosynthesis
• In bacteria
– allosterically inhibited at ATCase by CTP (or
UTP)
– allosterically activated at ATCase by ATP
(compete with CTP)
• In animals
– UDP and UTP are feedback inhibitors of CPS II
– PRPP and ATP are allosteric activators
Figure 26.17
A comparison of the regulatory circuits that control pyrimidine synthesis in E. coli and
animals.
26.6 – How Are Pyrimidines Degraded?
• In humans, pyrimidines are recycled from
nucleosides (via phosphoribosyltransferase),
but free pyrimidine bases are not salvaged
• Catabolism of cytosine and uracil yields balanine, ammonium, and CO2
– b-alanine can be recycled into the synthesis of
coenzyme A
• Catabolism of thymine yields baminoisobutyric acid, ammonium, and CO2
Figure 26.18
Pyrimidine degradation. Carbons
4, 5, and 6 plus N-1 are released
as b-alanine, N-3 as NH4+, and C-2
as CO2. (The pyrimidine thymine
yields b-aminoisobutyric acid.)
Recall that aspartate was the
source of N-1 and C-4, -5, and -6,
while C-2 came from CO2 and N-3
from NH4+ via glutamine.
26.7 – How Do Cells Form the
Deoxyribonucleotides That Are
Necessary for DNA Synthesis?
• Reduction at 2’-position of ribose ring
• Serve as precursor for DNA synthesis
• Replacement of 2’-OH with hydride is
catalyzed by ribonucleotide reductase
– An a2b2-type enzyme - subunits R1 (86 kD) and R2
(43.5 kD)
– R1 has two regulatory sites, a specificity site and
an overall activity site
Figure 26.19
Deoxyribonucleotide
synthesis involves
reduction at the 2'position of the ribose
ring of nucleoside
diphosphates.
Ribonucleotide Reductase
• The enzyme system consists of 4 proteins
– Two of which constitute the Ribonucleotide
Reductase (a2b2)
– Thioredoxin and thioredoxin reductase deliver
reducing equivalents
• Has three different nucleotide-binding sites
– Substrate: NDPs
– Activity-determining: ATP & dATP
– Specificity-determining: ATP, dTTP, dGTP, and
dATP
Figure 26.20
E. coli ribonucleotide reductase: its binding sites and subunit organization.
• Activity depends on Cys439, Cys225, and Cys462 on R1
and on Tyr122 on R2 (generate free radical)
• Tyr122 free radical on R2 leads to removal of the Ha
hydrogen (Cys439) and creation of a C-3‘ radical
• dehydration via removal of Hb together with the C-2‘OH group and restoration of Ha to C-3' forms the
dNDP product
• accompanied with disulfide formation between Cys225
and Cys462
• Thioredoxin provides the reducing power for
ribonucleotide reductase
• NADPH is the ultimate source
• Sulfide : sulfhydryl transition
Figure 26.22
The (SS)/(SH HS) oxidation-reduction cycle involving ribonucleotide
reductase, thioredoxin, thioredoxin reductase, and NADPH.
• Glutaredoxin can also function in ribonucleotide
reductase
• Oxidized Glutaredoxin is reduced by 2 Glutathione
Figure 26.23
The structure
of glutathione.
Regulation of dNTP Synthesis
• The overall activity of ribonucleotide
reductase must be regulated
– ATP activates, dATP inhibits at the overall
activity site
• Balance of the four deoxynucleotides must
be controlled
– ATP, dATP, dTTP and dGTP bind at the
specificity site to regulate the selection of
substrates and the products made
Figure 26.24
Regulation of deoxynucleotide biosynthesis: The rationale for the various affinities
displayed by the two nucleotide-binding regulatory sites on ribonucleotide reductase.
26.8 – How Are Thymine
Nucleotides Synthesized?
• Thymine nucleotides are made from dUMP, which
derives from dUDP, dCDP
• Thymidylate synthase methylates dUMP at 5position to make dTMP
• N5,N10-methylene THF is 1-C donor
• If the dCDP pathway is traced from the common
pyrimidine precursor, UMP, it will proceed as
follows:
UMP  UDP  UTP  CTP  CDP  dCDP  dCMP  dUMP  dTMP
Figure 26.26 The dCMP deaminase reaction.
Figure 26.27
The thymidylate synthase
reaction. The 5-CH3 group is
ultimately derived from the bcarbon of serine.
Figure 26.28
Precursors and analogs
of folic acid employed
as antimetabolites:
sulfonamides (see
Human Biochemistry
box on page 858), as
well as methotrexate,
aminopterin, and
trimethoprim, whose
structures are shown
here.
These compounds
shown here bind to
dihydrofolate reductase
(DHFR) with about
1000-fold greater
affinity than DHF and
thus act as virtually
irreversible inhibitors.
• Fluoro-substituted analogs as
therapeutic agents
The effect of the 5-fluoro substitution on the mechanism
of action of thymidylate synthase. An enzyme thiol
group (from a Cys side chain) ordinarily attacks the 6position of dUMP so that C-5 can react as a carbanion
with N5,N10-methylene-THF. Normally, free enzyme is
regenerated following release of the hydrogen at C-5 as
a proton. Because release of fluorine as F+ cannot occur,
the ternary (three-part) complex of [enzyme:
flourouridylate:methylene-THF] is stable and persists,
preventing enzyme turnover. (The N5,N10-methyleneTHF structure is given in abbreviated form.)
HB page 876
The structures of 5-fluorouracil
(5-FU),5-fluorocytosine, and 5fluoroorotate.