Download Chapter 26

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?
26.2 – How Do Cells Synthesize Purines?
• 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
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 26.3
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.
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-β-amine; Changes C-1
configuration (α→β)
– GMP and AMP inhibit this step - but at distinct sites
– Azaserine - Gln analog - inhibitor/anti-tumor
Figure 26.4 The structure of azaserine. Azaserine acts as an
irreversible inhibitor of glutamine-dependent 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 methotrexate and
sulfonamides block purine synthesis
Tetrahydrofolate and
One-Carbon Units
Tetrahydrofolate and One-Carbon
Units
Folic acid, a B vitamin found in
green plants, fresh fruits, yeast, and
liver, is named from folium, Latin for
“leaf”.
Folates are acceptors and donors of
one-carbon units for all oxidation
levels of carbon except CO2 (for
which biotin is the relevant carrier).
The active form is tetrahydrofolate.
Folates are acceptors and donors of one-carbon units for all
oxidation levels of carbon except CO2 (for which biotin is the
relevant carrier).
Tetrahydrofolate and One-Carbon Units
Oxidation numbers are calculated by assigning
valence bond electrons to the more
electronegative atom and then counting the
charge on the quasi ion. A carbon assigned four
valence electrons would have an oxidation
number of 0. The carbon in N5-methyl-THF (top
left) is assigned six electrons from the three C-H
bonds and thus has a oxidation number of -2.
Folate Analogs as Antimicrobial and
Anticancer Agents
• De novo purine biosynthesis depends on folic acid
compounds at steps 4 and 10
• For this reason, antagonists of folic acid metabolism
indirectly inhibit purine formation and, in turn, nucleic
acid synthesis, cell growth, and cell development
• Rapidly growing cells, such as infective bacteria and fastgrowing tumors, are more susceptible to such agents
• Sulfonamides are effective anti-bacterial agents
• Methotrexate and aminopterin are folic acid analogs that
have been used in cancer chemotherapy
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.
Figure 26.5 The synthesis
of AMP and GMP from
IMP.
AMP and GMP are Synthesized from IMP
Reciprocal control occurs in two ways - see
Figures 26.5 and 26.6
• GTP is the energy input for AMP synthesis,
whereas ATP is energy input for GMP
• AMP is made by N addition from aspartate (in
the familiar way - see Figure 26.5)
• GMP is made by oxidation at C-2, followed by
replacement of the O by N (from Gln)
• Last step of GMP synthesis is identical to the
first two steps of IMP synthesis
AMP and GMP are synthesized from
IMP
•
1.
–
–
–
2.
–
–
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
•
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
The regulation of purine synthesis
Reciprocal control occurs in two ways
IMP synthesis:
–
Allosterically regulated at the first two steps
AMP synthesis:
adenylosuccinate synthetase is feedback-inhibited by
AMP
GMP synthesis:
IMP dehydrogenase is feedback-inhibited by GMP
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
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
Victims of Lesch-Nyhan syndrome
experience severe arthritis due to
accumulation of uric acid, as well as
retardation, and other neurological
symptoms.
Hyperxanthine-Guanine PhosphoRibosylTransferase
Figure 26.7
Purine salvage by the HGPRT reaction.
Lesch-Nyhan syndrome
results from a complete
deficiency in HGPRT.
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)
Purines nucleotide cycle
• Serve as an anaplerotic pathway in skeletal
muscle
– AMP deaminase
– Adenylosuccinate synthetase
– Adenylosuccinate lyase
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.
Xanthine Oxidase and Gout
Figure 26.9 The purine
nucleoside cycle for
anaplerotic replenishment of
citric acid cycle
intermediates in skeletal
muscle.
• 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 xanthine oxidase ,
is a treatment
Animals other than humans
oxidize uric acid to form
excretory products
• Urate oxidase: Allantoin
• Allantoinase: Allantoic acid
• Allantoicase: Urea
• Urease: Ammonia
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.
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)
• 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
(also called carbonyl-phosphate)
Figure 26.14
The reaction catalyzed by
carbamoyl phosphate
synthetase II (CPS II).
• 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)
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
• The metabolic channeling is more efficient
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 βalanine, ammonium, and CO2
– β-alanine can be recycled into the synthesis of
coenzyme A
• Catabolism of thymine yields βaminoisobutyric acid, ammonium, and CO2
Figure 26.18 Pyrimidine degradation.
Carbons 4, 5, and 6 plus N-1 are released
as β-alanine, N-3 as NH4+, and C-2 as
CO2. (The pyrimidine thymine yields βaminoisobutyric 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 α2β2-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
(α2β2)
– 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.
• 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 follows with disulfide formation between
Cys225, and Cys462 and forms the dNDP product
• 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.
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 26.23
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 26.25 (a) The dCMP deaminase reaction. An alternative route to dUMP
is provided by dCDP, which is dephosphorylated to dCMP and then deaminated
by dCMP deaminase
Figure 26.26
The thymidylate
synthase reaction.
• Synthesis of dTMP from dUMP is catalyzed by
thymidylate synthase
• This enzyme methylates dUMP at the 5-position to
create dTMP
• The methyl donor is the one-carbon folic acid
derivative N5,N10-methylene-THF
• The reaction is a reductive methylation; the onecarbon unit is transferred at the methylene level of
reduction and then reduced to the methyl level
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.)
The structures of 5-fluorouracil (5FU),5-fluorocytosine, and 5fluoroorotate.