Transcript CHAPTER 6

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?
Nucleotides and Nucleic Acids
• Nucleotides: (nucleoside + phosphate)
– Biological molecules that possess a heterocyclic
nitrogenous base, a five-carbon sugar (ribose), and
phosphate as principal components (chapter 10)
– Participate as essential intermediates in cellular
metabolisms—NAD, FAD, ATP, cAMP…
– The elements of heredity and the agents of genetic
information transfer—nucleic acids
• Nucleic acids:
– Nucleotides are the monomeric units of nucleic acid
– Two basic kinds of nucleic acids
1.Deoxyribonucleic acid (DNA)
2.Ribonucleic acid (RNA)
26.1 – Can Cells Synthesize Nucleotides?
1. Nearly all organisms synthesize purines and
pyrimidines "de novo biosynthesis pathway“
2. Many organisms also "salvage" purines and
pyrimidines from diet and degradative
pathways
• Ribose can be catabolized to generate
energy, but nitrogenous bases 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 26.3 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
IMP (inosinic acid or inosine monophosphate) is
the immediate precursor to GMP and AMP
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 - Glutamine analog - inhibitor/anti-tumor
Figure 26.4 The structure of
azaserine. Azaserine acts as an
irreversible inhibitor of glutaminedependent enzymes by covalently
attaching to nucleophilic groups in
the glutamine-binding 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 (FGAM
synthetase)
– Formylglycinamidine ribonucleotide (FGAM)
– 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 (also see Fig 26.5)
– 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 are required in the purine biosynthesis
from ribose-5-phosphate to IMP, but that this
is really 7 ATP equivalents
• The dependence of purine biosynthesis on
THF (tetrahydrofolate) in two steps means that
methotrexate and sulfonamides block purine
synthesis
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.
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).
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.
AMP and GMP are Synthesized from IMP
Figure 26.5 The
synthesis of AMP
and GMP from
IMP.
AMP and GMP are synthesized from
IMP
IMP is the precursor to both AMP and GMP
• AMP synthesis
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
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
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
Nucleoside 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
• Nucleic acid turnover (synthesis and degradation) is
an ongoing metabolic process
– mRNA in particular is actively synthesized and
degraded
– Lead to release of free purines; adenine, guanine,
and hypoxanthine (the base in IMP; Fig 26.8)
• Salvage pathways exist to recover them in useful
form
• Involve resynthesis of nucleotides from bases via
phosphoribosyltransferases (PRT)
26.3 – Can Cells Salvage Purines?
Base + PRPP
Nucleoside-5’-phosphate + PPi
• The purine phosphoribosyltransferases are adenine
phosphoribosyltransferases (APRT) and
hypoxanthine-guanine phosphoribosyltransferases
(HGPRT)
• Collect hypoxanthine and guanine and recombine
them with PRPP to form nucleotides in the HGPRT
reaction (Fig 26.7)
– 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
Hyperxanthine-Guanine PhosphoRibosylTransferase
Figure 26.7
Purine salvage by the HGPRT reaction.
Victims of Lesch-Nyhan syndrome
experience severe arthritis due to
accumulation of uric acid, as well as
retardation, and other neurological
symptoms.
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.
The purine nucleoside cycle in skeletal muscle Serve as
an anaplerotic pathway
• Convert aspartate to fumarate plus NH4+
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 xanthine oxidase , 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 carbamoyl-aspartate
– 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 some organisms, free pyrimidines are
salvaged and recycled to form nucleotides
– In humans, pyrimidines are recycled from
nucleosides, 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?
• 90% of the total nucleic acid in cells is RNA,
with the remainder being DNA
• The deoxynucleotides have only one metabolic
purpose: to serve as precursor for DNA
synthesis
• NDPs are the substrate for deoxynucleotides
formation
– Reduction at 2’-position of ribose ring in NDPs
produces 2’-deoxy forms of these nucleotides
• Replacement of 2’-OH with hydride is catalyzed
by ribonucleotide reductase
Figure 26.19 Deoxyribonucleotide
synthesis involves reduction at the 2'position of the ribose ring of
nucleoside diphosphates.
Figure 26.20 E. coli ribonucleotide reductase.
• An a2b2-type enzyme
has subunits R1 (a2, 86
kD) and R2 (b2, 43.5
kD)
• R1 has two regulatory
sites, a substrate
specificity site (S) and
an overall activity site
(A)
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 (active site)
– Activity-determining: ATP & dATP (overall actiyity
site)
– Specificity-determining: ATP, dTTP, dGTP, and
dATP (sbustrate specific site)
• 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
• Reversible 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
1. The overall activity of ribonucleotide
reductase must be regulated
–
ATP activates, dATP inhibits at the overall
activity site
2. Balance of the four deoxynucleotides must
be controlled
–
ATP, dATP, dTTP and dGTP bind at the
substrate 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
dUTPase
dCMP deaminase
• Thymidylate synthase methylates dUMP at 5-position
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
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
• The THF cofactor is oxidized to yield DHF
• DHFR reduces DHF back to THF for serving again
• dTMP synthesis has become a preferred target for
inhibitors designed to disrupt DNA synthesis
Figure 26.26 The thymidylate
synthase reaction.
Precursors and analogs of folic acid employed as antimetabolites:
sulfonamides (see Human Biochemistry box on page 896), 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 6-position of dUMP so that
C-5 can react as a carbanion with N5,N10methylene-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-methylene-THF structure is given in
abbreviated form.)
5-fluorouracil (5-FU) is used as a
chemotherapeutic agent in the
treatment of human cancers
5-fluorocytosine is used as an antifungal
drug
5-fluoroorotate is an effective
antimalarial drug