Chapter 26 Synthesis and Degradation of Nucleotides

1
Chapter 26Synthesis and Degradation of
Nucleotides
2
Outline

• Nucleotide synthesis.
• Purine synthesis.
• Purine salvage pathway.
• Purines degradation.
• Pyrimidine synthesis.
• Pyrimidines degradation.
• Synthesis of deoxyribonucleotides that are
  necessary for DNA synthesis.
• Thymine nucleotide synthesis.

3
26.1 Can Cells Synthesize Nucleotides?

• Nearly all organisms synthesize purines and
  pyrimidines de novo (anew).
• 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.

4
26.2 Purine Synthesis

• John Buchanan (1948) "traced" the sources of all
  nine atoms of the purine ring
• N-1 from aspartic acid.
• N-3, N-9 from glutamine.
• C-4, C-5, N-7 from glycine.
• C-6 from CO2.
• C-2, C-8 from THF - one carbon units.

5
26.2 Purine Synthesis
Figure 26.2 The metabolic origin of the nine
atoms in the purine ring system.
6
26.2 Purines is a Waste Product in Birds
Figure 26.1 Nitrogen waste is excreted by birds
principally as the purine analog, uric acid.
7
26.2 Purine Synthesis, Steps 1 2

• The purine ring is built on a ribose-5-P
  foundation
• Step 1 Ribose-5-P must be activated - by PPi.
• Phosphoribosyl pyrophosphate (PRPP), made by PRPP
  synthetase, is the limiting substance for purine
  synthesis. (Used in Trp and His synthesis.)
• Since PRPP is a branch point, the next step is
  the committed step - Gln PRPP amidotransferase.
• Step 2 Note the change in C-1 configuration.
• G- and A-nucleotides inhibit this step - but at
  separate sites.

8
Purine Synthesis
Figure 26.3 The de novo pathway for purine
synthesis. The first purine synthesized is
inosine monophos. (IMP). IMP serves as a
precursor to AMP and GMP.
9
Purine Synthesis, Steps 3-5

• Step 3 Glycine carboxyl condenses with amine of
  5-phosphoribosyl-ß-amine in two steps.
• Glycine carboxyl activated by phosphorylation
  from ATP.
• Then the amine attacks the glycine carboxyl.
• Step 4 Formyl group of N10-formyl-THF is
  transferred to free amino group of GAR to form
  a-N-formylglycinamide ribonucleotide.
• Step 5 C-4 carbonyl forms a P-ester from ATP
  then active NH3 attacks C-4 to form an imine,
  namely formylglycinamidine ribonucleotide (FGAM).

10
Purine Synthesis
Figure 26.3 The de novo pathway for purine
synthesis.
11
Purine Synthesis, Steps 6-8

• Closure of the first ring, carboxylation and
  attack by aspartate
• Step 6 Similar in some ways to step 5. ATP
  activates the formyl group by phosphorylation,
  facilitating attack by N.
• Step 7 Carboxylation probably involves electron
  "push" from the amino group.
• Step 8 Attack by the amino group of aspartate
  links this amino acid with the carboxyl group.

12
Purine Synthesis
Figure 26.3 The de novo pathway for purine
synthesis.
13
Purine Synthesis, Steps 9-11

• Loss of fumarate, another 1-C unit and the second
  ring closure
• Step 9 Deprotonation of Asp-CH2 leads to
  cleavage to form fumarate.
• Step 10 Another 1-C addition catalyzed by THF.
• Step 11 Amino group attacks formyl group to
  close the second ring.
• Note that 6 steps use ATP, but that this is
  really seven ATP equivalents used to make IMP.

14
Purine Synthesis
Figure 26.3 This completes the de novo pathway
for purine synthesis. IMP is the first purine
made and serves as a precursor to AMP and GMP.
15
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.
16
Tetrahydrofolate and One-Carbon Units
Folates are acceptors and donors of one-carbon
units for all oxidation levels of carbon except
CO2 (for which biotin is the relevant carrier).
17
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.
18
Folate Analogs as Antimicrobial and Anticancer
Agents
19
AMP and GMP are Synthesized from IMP

• Reciprocal control occurs in two ways - see
  Figures 26.5 and 26.6
• GTP is the energy source for AMP synthesis, and
  ATP is the energy source for GMP.
• AMP is made by N addition from aspartate (in the
  familiar way).
• 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.

20
AMP is Made by Adenylosuccinate Lyase
AMP is made from IMP in two steps. The first
step converts IMP to adenylosuccinate and
requires GTP. The second step, catalyzed by
adenylosuccinate lyase produces AMP, as shown in
Figure 26.5. AMP synthesis from IMP requires one
additional ATP equivalent for a total of eight
ATPs.
21
AMP and GMP are Synthesized from IMP
Figure 26.5 The synthesis of AMP and GMP from
IMP.
22
GMP is Made by GMP Synthetase
GMP is made from IMP in two steps. The first
step requires oxidation at C-2 of the purine
ring. The second step, catalyzed by GMP
synthetase, is a glutamine-dependent
amidotransferase reaction that replaces the
oxygen on C-2 with an amino group to yield
2-amino, 6-oxy purine nucleoside monophosphate
i.e., GMP. GMP synthesis from IMP requires two
additional ATP equivalents for a total of nine
ATPs.
23
Formation of NDPs and NTPs
AMP and GMP are converted to NDPs using ATP and
nucleoside monophosphate kinase. ATP GMP ?
ADP GDP Conversion of GDP to GTP uses ATP
and nucleoside diphosphate kinase. ATP GDP
? ADP GTP
24
Regulation of the Purine Pathway
Figure 26.6 The regulatory circuit controlling
purine biosynthesis. Allosteric regulation occurs
in the first two steps, and AMP and GMP are
competitive inhibitors in the two branches at
right.
25
26.3 Purine Salvage Pathways

• Nucleic acid turnover (synthesis and degradation)
  is an ongoing process in most cells.
• Salvage pathways collect hypoxanthine and guanine
  and recombine them with PRPP to form nucleotides
  in the HGPRT reaction.
• (Hypoxanthine-guanine phosphoribosyltranferase)
  .
• In L-N, purine synthesis is increased 200-fold
  and uric acid is elevated in blood.
• This increase may be due to PRPP feed-forward
  activation of de novo pathways.

26
HGPRT Converts Bases Back to Nucleotides Using
PRPP
Salvage pathways recover purine bases to use for
resynthesis of nucleotides. e.g. the HGPRT
reaction will recover hypoxanthine or guanine.
Figure 26.7 Purine salvage by the HGPRT
reaction. HGPRT salvage of hypoxanthine. SN2
gives a ß-link.
27
HGPRT Converts Bases Back to Nucleotides Using
PRPP
Salvage pathways are very useful because of the
high energy cost for denovo synthesis of nitrogen
bases. The salvage pathway for adenine
recovery (adenine phosphoribosyltranferase) is
not shown.
Figure 26.7 Purine salvage by the HGPRT
reaction. HGPRT salvage of guanine. SN2 gives a
ß-link.
28
Some Commonly Used Enzymes

• Nucleotidases cleave Pi from a nucleotide.
• Nucleosidases cleave the base from a nucleoside.
• Nucleoside phosphorylase cleaves the base from a
  nucleoside using Pi.
• Nucleoside kinase adds phosphate to a nucleoside.

29
26.4 Purine Catabolism

• Purine catabolism leads to uric acid
• (see Figure 26.8)
• Nucleotidases and nucleosidases release ribose
  and phosphates and leave free bases.
• Xanthine oxidase and guanine deaminase route
  everything to xanthine.
• Xanthine oxidase converts xanthine to uric acid.
• Note that xanthine oxidase can oxidize two
  different sites on the purine ring system.

30
The Major Pathways of Purine Catabolism Lead to
Uric Acid

• Nucleotides are first converted to nucleosides
  (loss of Pi) by intracellular nucleotidases.
• These nucleotidases are under strict regulation
  to ensure that their substrates (which are
  intermediates in many processes) are not depleted
  below critical levels.
• Nucleosides are then degraded by purine
  nucleoside phosphorylase (PNP) giving ribose-1-P
  and a nitrogen base.
• PNP products are converted to xanthine by guanine
  deaminase and xanthine oxidase.

31
The Major Pathways of Purine Catabolism Lead to
Uric Acid
Figure 26.8 The major pathways for purine
catabolism.
32
The Major Pathways of Purine Catabolism Lead to
Uric Acid
Figure 26. The major pathways for purine
catabolism.
33
The Purine Nucleotide Cycle in Skeletal Muscle
Serves as an Anaplerotic Pathway

• Deamination of AMP to IMP by AMP deaminase,
  followed by resynthesis of AMP from IMP in de
  novo purine pathway enzymes, constitutes a purine
  nucleoside cycle (Figure 26.9).
• This cycle has the net effect of converting
  aspartate to fumarate plus NH4.
• This nucleotide cycle is important fumarate
  that is generated replenishes the levels of
  citric acid intermediates lost in amphibolic side
  reactions.
• Skeletal muscle lacks the usual anaplerotic
  enzymes and relies on AMP deaminase for this
  purpose.

34
The Purine Nucleotide Cycle in Skeletal Muscle
Serves as an Anaplerotic Pathway
Figure 26.9 The purine nucleoside cycle for
anaplerotic replenishment of citric acid cycle
intermediates in skeletal muscle.
35
Gout is a Disease Caused by an Excess of Uric Acid

• Xanthine oxidase (XO) in liver, intestines (and
  milk) can oxidize hypoxanthine (twice) 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.

36
Gout is a Disease Caused by an Excess of Uric Acid
Figure 26.10 Xanthine oxidase catalyzes a
hydroxylase-type reaction.
37
Gout is a Disease Caused by an Excess of Uric Acid
Figure 26.11 Allopurinol, an analog of
hypoxanthine, is a potent inhibitor of xanthine
oxidase. Allopurinol binds tightly to xanthine
oxidase, preventing uric acid formation.
Hypoxanthine and xanthine do not accumulate to
harmful concentrations because they are more
soluble and thus more easily excreted.
38
Animals Oxidize Uric Acid to Different Excretory
Products
Figure 26.12 The catabolism of uric acid to
allantoin, allantoic acid, urea, or ammonia in
various animals. Primates, like birds, reptiles,
and insects, excrete uric acid directly. Other
animals use other routes of excretion.
39
Animals Oxidize Uric Acid to Different Excretory
Products
Figure 26.12 The catabolism of uric acid to
allantoin, allantoic acid, urea, or ammonia in
various animals. Primates, like birds, reptiles,
and insects, excrete uric acid directly. Other
animals use other routes of excretion.
40
26.5 Pyrimidine Synthesis

• In contrast to purines, pyrimidines are not
  synthesized as nucleotides.
• Rather, the pyrimidine ring is completed before a
  ribose-5-P is added.
• Carbamoyl-phosphate and aspartate are the
  precursors of the six atoms of the pyrimidine
  ring.
• Mammals have two enzymes for carbamoyl phosphate
  synthesis carbamoyl phosphate for pyrimidine
  synthesis is formed by carbamoyl phosphate
  synthetase II (CPS-II), a cytosolic enzyme.

41
26.5 Pyrimidine Synthesis
Figure 26.13 The metabolic origin of the six
atoms of the pyrimidine ring. Only two precursors
contribute atoms to the six-membered pyrimidine
ring.
42
Carbamoyl Phosphate Synthetase II

• Step1 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, 2 ATP.
• See Figure 27.14.
• Mammalian CPS II is the committed step in
  pyrimidine synthesis, because carbamoyl phosphate
  made by CPS II has no fate other than
  incorporation into pyrimidines.

43
Carbamoyl Phosphate Synthetase II
Figure 26.14 The carbamoyl phosphate synthetase
II (CPS II) reaction. This is the committed
step in pyrimidine synthesis in mammals.
44
Carbamoyl Phosphate Synthetase II
Figure 26.14 The carbamoyl phosphate synthetase
II (CPS II) reaction.
45
Step 2 Aspartate transcarbamoylase

• Step 2 Aspartate transcarbamoylase (ATCase)
  catalyzes the condensation of carbamoyl phosphate
  with aspartate to form carbamoyl-aspartate.
• Note that carbamoyl phosphate represents an
  activated carbamoyl group.
• Aspartate transcarbamoylase is a classic
  two-state allosteric enzyme (ATP, CTP-).

46
ATCase Converts Carbamoyl Phosphate to Carbamoyl
Aspartate
Figure 26.15 The de novo pyrimidine biosynthetic
pathway.
47
Steps 3-6 of Pyrimidine Biosynthesis

• Step 3 ring closure and dehydration - catalyzed
  by dihydroorotase.
• Step 4 Synthesis of a true pyrimidine (orotate)
  by DHO dehydrogenase.
• Step 5 Orotate is joined with a ribose-P to form
  orotidine-5'-phosphate.
• The ribose-P donor is PRPP.
• Step 6 OMP decarboxylase makes UMP.
• These are six separate enzymes in procaryotes and
  three multifunctional proteins in eucaryotes.

48
Metabolic Channeling by Multifunctional Enzymes
of Pyrimidine Biosynthesis

• Eukaryotic pyrimidine synthesis involves
  metabolic channeling (the transfer of metabolites
  between different enzymatic sites on a
  multifunctional polypeptide).
• ATP requirements for denovo pyrimidine synthesis
• To UMP 4 ATP
• To CTP 5 ATP (not counting UDP UTP)
• CTP synthetase then forms CTP from UTP and ATP.

49
UMP Synthesis Leads to Formation of UTP and CTP
Figure 26.16(a) CTP synthesis from UTP. UDP is
formed from UMP via an ATP-dependent nucleoside
monophosphate kinase UMP ATP ? UDP ADP Then
UTP is formed by nucleoside diphosphate
kinase UDP ATP ? UTP ADP
50
Pyrimidine Biosynthesis is Regulated at ATCase in
Bacteria and at CPS II in Animals
Figure 26.17 A comparison of the regulatory
circuits that control pyrimidine synthesis in E.
coli and animals.
51
26.3 Pyrimidine Salvage Pathways
PYRIMIDINES uracil Uracil
PRPP --------------gt UMP PPi
phosphoribosyl transferase Orotate
orotate Uracil PRPP ----------------gt
PyMP PPi Cytosine
phosphoribosyl transferase
52
26.6 Pyrimidines Degradation

• In some organisms, free pyrimidines are salvaged
  and recycled to form nucleotides via
  phosphoribosyltransferase reactions.
• In humans, however, pyrimidines are recycled from
  nucleosides, but free pyrimidine bases are not
  salvaged.
• Catabolism of cytosine and uracil yields
  ?-alanine, ammonium ion, and CO2.
• Catabolism of thymine yields ?-aminoisobutyric
  acid, ammonium ion, and CO2.

53
26.6 Pyrimidines Degradation
Figure 26.18 Pyrimidine degradation. Catabolism
of cytosine and uracil yields ?-alanine, ammonium
ion, and CO2. Catabolism of thymine yields
?-aminoisobutyric acid, ammonium ion, and CO2.
54
26.7 Synthesis of Deoxyribonucleotides

• Reduction of an NDP at the 2'-position by
  replacement of the 2'-OH with hydride commits
  nucleotides to DNA synthesis.
• This reduction with hydride is catalyzed by
  ribonucleotide reductase.
• Ribonucleotide reductase is an ?2?2-type enzyme -
  subunits R1 (86 kD) and R2 (43.5 kD).
• Each R1 subunit has regulatory site, a
  specificity site and an overall activity site.
  Each R2 subunit has part of the activity site.

55
26.7 Synthesis of Deoxyribonucleotides
Figure 26.19 Deoxyribonucleotide synthesis
involves reduction at the 2'-position of the
ribose ring of nucleoside diphosphates.
56
E. Coli Ribonucleotide Reductase Has Three
Different Nucleotide-Binding Sites
Figure 26.20 E. coli ribonucleotide reductase.
57
Ribonucleotide Reductase is Regulated by
Nucleotide Binding

• Ribonucleotide reductase is modulated in two ways
  to maintain a balance of dATP, dGTP, dCTP, and
  dTTP.
• First, the catalytic activity of the enzyme of
  the enzyme must be turned on and off in response
  to need for dNTPs.
• Second, the amounts of each NDP substrate
  transformed must be controlled.
• ATP activates, dATP inhibits at the overall
  activity site.
• ATP, dATP, dTTP and dGTP bind at the specificity
  site to regulate the selection of substrates and
  the products made.

58
E. Coli Ribonucleotide Reductase

• Effects of binding at S, A and C
• Bound at A Bound at S Accepted at C
• ATP (on) ATP or dATP CDP or UDP
• ATP (on) dTTP GDP favored
• CDP or UDP (no)
• ATP (on) dGTP ADP favored
• CDP, UDP, GDP (no)
• dATP (off) Any No reaction

59
Ribonucleotide Reductase is Regulated by
Nucleotide Binding
Figure 26.23 Regulation of deoxynucleotide
biosynthesis the rationale for the various
affinities displayed by the two
nucleotide-binding regulatory sites on
ribonucleotide reductase.
60
E. Coli Ribonucleotide Reductase Has Three
Different Nucleotide-Binding Sites

• Activity depends on Cys439, Cys225, and Cys462 on
  R1 and on Tyr122 on R2.
• Cys439 removes 3'-H, and dehydration follows,
  with disulfide formation between Cys225 and
  Cys462.
• The net result is hydride transfer to C-2.
• Thioredoxin and thioredoxin reductase deliver
  reducing equivalents.

61
Ribonucleotide Reductase Uses a Free Radical
Mechanism
Figure 26.21 The free radical mechanism of
ribonucleotide reduction. Ha designates the C-3'
hydrogen and Hb the C-2' hydrogen atom.
62
Thioredoxin Reductase Mediates NADPH-Dependent
Reduction of Thioredoxin
Figure 26.22(a) The oxidation-reduction cycle
involving ribonucleotide reductase, thioredoxin,
thioredoxin reductase, and NADPH. Thioredoxin
functions in a number of metabolic roles besides
deoxyribonucleotide synthesis.
63
26.8 Synthesis of Thymine Nucleotides

• Thymine nucleotides are made from dUMP, which
  derives from dUDP, dCDP.
• dUDP?dUTP?dUMP?dTMP.
• dCDP?dCMP?dUMP?dTMP.
• Thymidylate synthase methylates dUMP at
  5-position to make dTMP.
• N5,N10-methylene THF is 1-C donor.
• Note role of 5-FU in chemotherapy.

64
26.8 Synthesis of Thymine Nucleotides
Figure 26.24 Pathways of dTMP synthesis. dTMP
production is dependent on dUMP formation from
dCDP and dUDP. Interestingly, formation of dUMP
from dUDP passes through dUTP, which is then
cleaved by dUTPase, a pyrophosphatase that
removes PPi from dUTP. The action of dUTPase
prevents dUTP from serving as a substrate in DNA
synthesis.
65
dCMP Deaminase Provides an Alternative Route to
dUMP
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.
66
26.8 Synthesis of Thymine Nucleotides

• 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
  one-carbon unit is transferred at the methylene
  level of reduction and then reduced to the methyl
  level.

67
Thymidylate Synthase
Figure 26.26(a) The thymidylate synthase
reaction.
68
End Chapter 26Synthesis and Degradation of
Nucleotides