17,Amino acids are precursors of
many specialized biomolecules
? Radioisotope tracer experiments showed that the
porphyrin (卟啉 ) ring of the heme groups in
mammals are made from 8 Gly and 8 succinyl-CoA
(Glu in plants and bacteria).
? Deficiency of enzymes leading to heme biosynthesis
cause diseases called porphyrias,
? (The degradation of heme yields bilirubin,a linear
tetrapyrrole derivative,and the typical pigments found
in bile,urine,and feces,a deficiency of which will
cause jaundice,黄疸 ).
? Creatine,the ―energy buffer‖ in skeletal muscle,is
made from Gly,Arg,and Met.
? Glutathione,the ―redox buffer‖,is derived from Glu,
Cys,and Gly.
? Indole-3-acetate (or auxin),the plant growth
hormone,is derived from Trp.
? Many important neurotransmitters,e.g.,g-
aminobutyrate (GABA),serotonin (5-
hydroxytrptamine,or 5-HT,5-羟色胺 ),dopamine,
norepinephrine,epinephrine are derived from amino
acids via simple pathways,all include a PLP-
depedent decarboxylation reaction,
? Polyamines (e.g.,spermidine and spermine),needed
for DNA packaging,is made from Met and ornithine.
? Nitric oxide (NO),a stable free radical signals for
vasodilation (血管舒张 ) in vertebrates,is made
from the guanidino group of Arg and O2 in a
reaction catalyzed by an monooxygenase,nitric
oxide synthase (NOS).
? NOS is a dimeric enzyme with each subunit
containing an array of redox coenzymes,NADP,
FMN,FAD,tetrahydrobiopterin,and Fe3+-heme.
Porphyrins
In mammals are
made from Gly
and succinyl-CoA
(reactions 1 and 6
occur in the mitochondria,
the rest in the cytosols)
d-aminolevulinate
synthase (main
control target for
heme biosynthesis)
胆色素原
原卟啉
d-氨基 —g-酮戊酸
Heme
Ferrochelatase
亚铁螯合酶
Fe2+
Creatine (肌酸 ),the,energy
buffer” in skeletal muscle,
is derivded from Gly,Arg,
and Met.
Glutathione is
made from Glu,
Cys,and Gly
GSH is probably important in
maintaining proteins in their reduced
forms.
?GSH is an important cofactor for
glutathione peroxidase,an enzyme
containing an essential selenocysteine
(硒代半胱氨酸 ) residue in the active
site,to remove toxic peroxides in cells.
How is glutathione (GSH) synthesized?
? GSH,having the structure of g-Glu-Cys-Gly,is the
redox buffer in cells,cycling between GSH
(reduced form) and GSSG (oxidized form);
? The tripeptide is synthesized using specific
enzymes (not ribosomes)
The plant growth hormone,
indole-3-acetate,is derived
from Trp.
Many neurotransmitters are
derived from amino acids
Spermidine and spermine
are derived from Met
and ornithine.
Nitric oxide (NO) is made from Arg and O2 in a reaction
catalyzed by nitric oxide synthase (NOS),a
monooxygenase containing five coenzymes,NADP,FMN,
FAD,tetrahydrobiopterin,and a Fe 3+-heme,
(Robert F,Furchgott,Louis J,Ignarro,and Ferid Murad were
awarded the 1998 Nobel Prize in Medicine for revealing the
role of NO in the cardiovascular systems)
18,Nucleotides are synthesized via
either the de novo pathways or the
salvage pathways
? In the de novo pathway simple precursors,
including amino acids,PRPP,NH4+,CO2,
and one-carbon units (carried on H4
folate) are used.
? In the salvage pathway the free bases and
nucleosides released from nucleic acid
breakdown are used.
? The free bases (A,G,C,U,T) are not intermediates
during the de novo synthesis,the purine ring is
assembled on ribose phosphate to make AMP and
GMP; the pyrimidine ring is first synthesized as
orotate,which is then attached to ribose phosphate
before being converted to UTP and CTP (dTMP is
made from dUMP).
? The deoxyribonucleotides (dNDPs) are synthesized
by reduction of ribonucleotides (NDPs).
19,Radioisotope tracer experiments
revealed the origins of the atoms in
the purine and pyrimidine rings
? Buchanan and Greenberg revealed this by feeding a
variety of isotopically labeled compounds to pigeons
(1940s).
? The atoms of the purine rings were found to be
derived from formate,CO2,Gly,Asp,and Gln.
? The atoms of the pyrimidine rings were found to be
derived from Asp,Gln and HCO3-.
Radioisotope tracer experiments revealed
the origins of the ring atoms of purines
Gln amide
HCO3-
C
The atoms of the pyrimidine rings were revealed to
Be derived from HCO3-,Gln and Asp.
20,De novo purine nucleotide
synthesis begins with the transferring
of an amino group from Gln to PRPP
? PRPP is synthesized from ribose 5-P.
? In the first (committing) step of purine nucleotide synthesis,
the PPi part on PRPP (at C-1) is replaced by the side chain
amino group of Gln,forming 5-phosphoribosylamine.
? A glycine (2C +1N),a N10-formyl H4 folate (1C),a Gln
(1N),a HCO3-(1C) Asp (1N),and another N10-formyl H4
folate (1C) then brings the rest atoms for the purine ring.
? The first intermediate having a complete purine ring
is IMP (inosinate).
? IMP is converted to AMP by accepting an amino
group from Asp,and converted to GMP by a
NAD+-dependent dehydrogenation reaction (at C-2)
and an amino group transfer from Gln (catalyzed by
an amidotransferase).
? The production of AMP from IMP requires GTP,
and the production of GMP from IMP requires ATP.
PRPP is synthesized from ribose 5-P in a
Reaction catalyzed by PRPP synthetase.
The purine ring is built up one or a few atoms at
a time on ribose 5-P
Steps 1,3,and 5 are
catalyzed by one
multifunctional protein
in some eukaryotic cells.
IMP is first
formed and
is then converted
to AMP and
GMP.
Steps 7 and 8 are
catalyzed by one
protein in some
eukaryotic cells
Steps 10 and 11 are
catalyzed by one protein
in some eukaryotic cells
IMP is converted to AMP by accepting an amino
group from Asp (GTP is needed to activate Asp) and to
GMP by accepting an amino group from Gln (ATP is
needed to activate XMP)
21,The biosynthesis of AMP and
GMP is regulated by feedback
inhibition
PRPP synthetase and glutamine-PRPP
amidotransferase are both inhibited by the end
products IMP,AMP and GMP (AMP and
GMP act synergistically).
AMP inhibits adenylosuccinate synthetase and
GMP inhibits IMP dehydrogenase.
AMP and GMP synthesis is balanced by the
following mechanism,GTP is needed for
AMP synthesis and ATP for GMP synthesis.
The de novo synthesis
of AMP and GMP
is regulated mainly
by sequential feedback
Inhibition.
22,The de novo biosynthesis of
pyrimidines begin with the formation
of carbamoyl phosphate
? Carbamoyl phosphate is formed from Gln,HCO3-,
and ATP in a reaction catalyzed by the carbamoyl
phosphate synthetase II.
? In the committing step of pyrimidine biosynthesis,
carbamoyl phosphate condenses with Asp to form
N-carbamoylaspartate in a reaction catalyzed by
aspartate transcarbamoylase (ATCase).
? A ring closing reaction generates L-dihydroorotate,
which is then oxidized to form orotate (乳清酸 ) in a
reaction catalyzed by dihydroorotate dehydrogenase.
? Orotate is then linked to PRPP to form orotidylate,
which is then converted to UMP by a decarboxylation
reaction.
? CTP is derived from UTP by accepting an amino
group from Gln or NH4+.
? In eukaryotic cells,carbamoyl phosphate synthetase
II,aspartate transcarbamoylase,dihydroorotase,and
are part of a trifunctional proteins called CAD.
? When PALA,an ATCase inhibitor was added
to cultured mammalian cells,concentrations of
all three enzymes are increased dramatically
for cells resistant to PALA.
Gln
HCO3-+ATP
NH4+
Carbamoyl phosphate
Intermediates in
bacterial carbamoyl
phosphate synthetase
is channeled.
23,Pyrimidine nucleotide biosynthesis
is regulated at the aspartate
transcarbamoylase (ATCase)
? ATCase is inhibited by CTP,the end product of the
de novo pyrimidine nucleotide biosynthesis pathway.
? CTP binds at the regulatory subunits,which changes
the conformation of the catalytic subunits,
inactivating them.
? ATP is able to prevent the changes induced by CTP.
ATCase contains separate catalytic and
regulatory subunits
Catalytic
Subunits (binding Asp and carbamoyl phosphate)
Regulatory subunits (binding CTP or ATP)
24,Base specific NMP kinases
together with a nonspecific NDP
kinase converts NMPs to NTPs
? Nucleotides participate in biosynthesis (of RNA and
DNA) in the forms of NTPs.
? Each specific nucleoside monophosphate (NMP)
kinase converts the corresponding NMP/dNMPs to
NDP/dNDPs using ATP.
? The nonspecific nucleoside diphosphate (NDP)
kinase converts all NDP/dNDPs to NTP/dNTPs
using ATP or other NTPs.
25,Deoxyribonucleotides are made
from ribonucleotides at the NDP
level
? This occurs by direct reduction at the 2`-carbon.
? Ribonucleotide reductase catalyzes all such
conversions.
? The electrons are provided by NADPH and is
transferred to the ribonucleotide reductase via two
possible paths (via either thioredoxin or
glutaredoxin).
NDPs are converted
to dNDPs with the
catalysis of
ribonucleotide
reductase,with
electrons ultimately
coming from NADPH
Electrons are transferred from NADPH to the ribonucleotides
Via thioredoxin
(硫氧还蛋白 ）
Via glutaredoxin
(谷氧还蛋白 ）
26,Free radicals are involved in
converting NDPs to dNDPs by the
ribonucleotide reductase
? Ribonucleotide reductase exists as a tetramer,with
two R1 and two R2 subunits.
? Each tetramer has two active sites formed at the
interface of R1 and R2 subunits,R1 contributes two –
SH groups and R2 contribute a radical (stabilized by
a Tyr radical,which,in turn is stabilized by a Fe3+-
Fe3+ binuclear center in class I ribonucleotide
reductase),
? The deoxygenation reaction is proposed to
occur via a 3`-ribonucleotide radical,which
help stabilize the 2` carbon cation
subsequently formed.
Ribonucleotide
reductase is a
tetramer of two
different subunits
The R2 dimer of
ribonucleotide reductase,
with a Tyr radical and a
Fe3+-Fe3+ binuclear center.
Tyr radical
Fe3+-Fe3+
A proposed
mechanism for
converting a
NDP to a dNDP
by ribonucleotide
reductase,the
radical property
of the enzyme is
transiently
transferred to the
substrate; the
presence of the
radical at C-3
stabilizes the
carbon cation
formed at C-2.
27,The E,coli ribonucleotide
reductase is regulated for both its
activity and substrate specificity
? Two types of regulatory sites are present on the R1
subunits,one for substrate specificity and the other
for overall enzyme activity.
? At the substrate specificity site,when dATP or ATP
binds,reduction of CDP and UDP is favored; when
dTTP binds,reduction of GDP is favored (and the
reduction of CDP and UDP is inhibited); when
dGTP binds,reduction of ADP is favored.
? At the overall enzymatic activity site,when
ATP binds,the enzyme is activated; when
dATP binds,the enzyme is inactivated.
Both the substrate specificity and
the overall enzymatic activity of
ribonucleotide reductase is
regulated to balance the
biosynthesis of all nucleotides.
28,dTMP is synthesized by
methylation of dUMP
? dUTP is first formed from either dUDP (via
phosphorylation) or dCTP (via deamination).
? dUMP is then formed from dUTP in a reaction
catalyzed by dUTPase (keeping dUTP at a low level
to prevent its incorporation into DNA).
? dUMP is then converted to dTMP by the catalysis of
thymidylate synthase,with a methylene group
transferred from and reduced by N5,N10-methylene
H4 folate (being donors of both one-carbon unit and
electrons!).
? The dihydrofolate is reduced to H4 folate by
NADPH in a reaction catalyzed by dihydrofolate
reductase (DHFR).
? The N5,N10-methylene H4 folate is regenerated from
Ser and tetrahydrofolate in a reaction catalyzed by
serine hydroxymethyltransferase.
? The reactions catalyzed by ribonucleotide
reductase and thymidylate synthase are probably
key for the transition from an RNA world to one in
which DNA stores genetic information.
dTMP is derived from dUMP via a methylation
reaction using N5,N10-methylene H4 folate as
donors of both one-carbon unit and electrons.
dUMP is converted to
dTMP in a N5,N10-
methylene H4 folate-
dependent reaction
catalyzed by
thymidylate synthase.
29,Uric acid is the excreted end
product of purine catabolism in
humans and many other animals
? For adenosine,the amino group is hydrolyzed before
the ribose group is removed.
? For guanosine,the amino group is hydrolyzed after
the ribose group is removed.
? Xanthine oxidase,having multiple cofactors
(including an FAD,a Mo complex,four different
Fe-S clusters),catalyzes the O2-dependent
conversion of hypoxanthine to xanthine and
xanthine to uric acid.
? Uric acid can be further converted to allantoin,
allantoate,urea or NH4+ in various animals.
? The deficiency of adenosine deaminase causes the
severe immunodeficiency disease in humans (it is
likely the accumulated adenosine is converted to
dATP,which inhibits the formation of all dNDPs by
ribonucleotide reductase).
? Overproduction of uric acid was revealed to cause
gout (痛风 ).
? Allopurinol,an inhibitor of xanthine oxidase,is used
to treat gout.
(黄嘌呤 )
(尿酸 )
(尿囊素 ）
(尿囊酸 )
Dephosphorylation
Dephosphorylation
Deamination
Deamination
DeribosylationDeribosylation
Oxidation
Oxidation
Allopurinol was designed to be a competitive inhibitor of
xanthine oxidase to treat gout by Elion and Hitchings,who
shared the Nobel Prize in 1988 for their discoveries of
important principles for drug treatment
30,Pyrimidines are broken down via
reduction
? The degradation of thymine produces
mathylmalonyl-CoA,which can be converted to
succinyl-CoA (a citric acid cycle intermediate) by
the catalysis of a mutase.
? The degradation of uracil and cytidine produces
malonyl-CoA,which is one precursor for fatty acid
biosynthesis.
? To a limited extent,catabolism of pyrimidine
nucleotides contributes to the energy metabolism of
the cell.
Propionyl-CoA Succinyl-CoA
Degradation products of
pyrimdines can enter the
citric acid cycle
31,Purine and pyrimidine bases can
be reconverted into nucleotides via
the salvage pathway
? Adenine phosphoribosyltransferase catalyzes the synthesis
of AMP from adenine and PRPP.
? Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
catalyzes the synthesis of GMP and IMP.
? The lack of HGPRT will cause Lesch-Nyhan syndrome.
? Pyrimidine bases are recycled in a similar way in
microorganisms,but pyrimidine bases does not seem to be
salvaged in significant amounts in mammals.
The purine bases can be converted
to purine nucleotides via the
salvage pathways
32,Many cancer chemotherapeutic
drugs target enzymes in the
nucleotide biosynthetic pathways
? Analogs of Gln,like azaserine and acivicin,inhibits many
amidotransferases used in nucleotide (and amino acid)
biosynthesis.
? Fluorouracil,after being converted to FdUMP by the
salvage pathway,can inhibit the thymidylate synthase after
two steps of conversion,thus inhibit the dTMP synthesis.
? Methotrexate,a folate analog,inhibits the dihydrofolate
reductase,thus the dTMP synthesis.
Azaserine and acivicin
inhibits amidotransferases,
thus inhibit the biosynthesis
of nucleotides and amino
acids.
FdUMP (from fluorouracil)
and methotrexate inhibits
thymidylate synthase and
DHFR respectively.
FdUMP is an suicide inhibitor of
thymidylate synthase
Summary
? Atmospheric N2 is reduced to ammonia by
the dinitrogenase reductase and the
dinitrogenase (containing a key Fe-Mo
cofactor) of the nitrogenase complex present
only in certain bacteria.
? Ammonia enters organic molecules via Glu
and Gln.
? Glutamine amidotransferases catalyzes the
transferring of the amide amino group to
many acceptor molecules.
? Amino acids are mainly derived from intermediates
of glycolysis,the citric acid cycle,and the pentose
phosphate pathway.
? Pro and Arg are derived from Asp,which is
synthesized from a-ketoglutarate.
? Ser,Gly,and Cys are derived from 3-
phosphoglycerate.
? Lys is derived from oxaloacetate (as Asp),pyruvate
and Glu; Met is derived from oxaloacetate (as Asp),
Cys,and N5-methyl H4 folate; Thr is derived from
oxaloacetate.
? Ile and Val are derived from Thr/pyruvate and two
molecules of pyruvate respectively,using the same
enzymes; Leu is derived from two molecules of
pyruvate,sharing four steps of reactions with Val
synthesis.
? Trptophan is derived from part of the atoms of two
molecules of phosphoenolpyruvate,one erythrose 4-
P,one GLn,one PRPP,and one Ser; Phe and Tyr
are synthesized from two phosphoenolpyruvates,
one erythrose 4-P,and one Glu.
? His is derived from one PRPP,one ATP,One Gln,
and one Glu.
? Amino acid biosynthesis is regulated by various
forms of feedback inhibition (including enzyme
multiplicity,concerted inhibition and sequential
feedback inhibition).
? Many other biomolecules (including hemes,creatine,
glutathione,plant growth hormones,
neurotransmitters,polyamies,and nitric oxide) are
derived from amino acids.
? Purine nucleotides are synthesized from PRPP,Gln,
Gly,N10-formyl H4 folate,Gln,HCO3-,Asp through
the de novo pathway.
? Pyrimidine nucleotides are synthesized using HCO3-,
Gln,Asp,and PRPP.
? De novo synthesis of nucleotides are regulated via
feedback inhibition (no covalent modifications yet
revealed).
? Deoxyribonucleotides are derived from
ribonucleotides at the NDP level,with the catalysis
of ribonucleotide reductase,which contains a chain
of electron carriers,uses free radicals,and being
regulated for both substrate specificity and overall
enzymatic activities.
? The dTMP molecule is derived from dUMP by
thymidylate synthase,an enzyme using N5,N10-
methylene-tetrahydrofolate as the donor of both one-
carbon unit and electrons.
? Degradation of purines and pyrimidines produces uric
acid and citric acid cycle intermediate/fatty acid
synthesis precursor,respectively.
? Purine and pyrimidine bases can be reused via the
salvage pathway.
? Many cancer chemotherapeutic drugs (e.g.,azaserine,
acivicin,fluorouracil,and methotrexate) inhibits
enzymes in the nucleotide biosynthetic pathways.
many specialized biomolecules
? Radioisotope tracer experiments showed that the
porphyrin (卟啉 ) ring of the heme groups in
mammals are made from 8 Gly and 8 succinyl-CoA
(Glu in plants and bacteria).
? Deficiency of enzymes leading to heme biosynthesis
cause diseases called porphyrias,
? (The degradation of heme yields bilirubin,a linear
tetrapyrrole derivative,and the typical pigments found
in bile,urine,and feces,a deficiency of which will
cause jaundice,黄疸 ).
? Creatine,the ―energy buffer‖ in skeletal muscle,is
made from Gly,Arg,and Met.
? Glutathione,the ―redox buffer‖,is derived from Glu,
Cys,and Gly.
? Indole-3-acetate (or auxin),the plant growth
hormone,is derived from Trp.
? Many important neurotransmitters,e.g.,g-
aminobutyrate (GABA),serotonin (5-
hydroxytrptamine,or 5-HT,5-羟色胺 ),dopamine,
norepinephrine,epinephrine are derived from amino
acids via simple pathways,all include a PLP-
depedent decarboxylation reaction,
? Polyamines (e.g.,spermidine and spermine),needed
for DNA packaging,is made from Met and ornithine.
? Nitric oxide (NO),a stable free radical signals for
vasodilation (血管舒张 ) in vertebrates,is made
from the guanidino group of Arg and O2 in a
reaction catalyzed by an monooxygenase,nitric
oxide synthase (NOS).
? NOS is a dimeric enzyme with each subunit
containing an array of redox coenzymes,NADP,
FMN,FAD,tetrahydrobiopterin,and Fe3+-heme.
Porphyrins
In mammals are
made from Gly
and succinyl-CoA
(reactions 1 and 6
occur in the mitochondria,
the rest in the cytosols)
d-aminolevulinate
synthase (main
control target for
heme biosynthesis)
胆色素原
原卟啉
d-氨基 —g-酮戊酸
Heme
Ferrochelatase
亚铁螯合酶
Fe2+
Creatine (肌酸 ),the,energy
buffer” in skeletal muscle,
is derivded from Gly,Arg,
and Met.
Glutathione is
made from Glu,
Cys,and Gly
GSH is probably important in
maintaining proteins in their reduced
forms.
?GSH is an important cofactor for
glutathione peroxidase,an enzyme
containing an essential selenocysteine
(硒代半胱氨酸 ) residue in the active
site,to remove toxic peroxides in cells.
How is glutathione (GSH) synthesized?
? GSH,having the structure of g-Glu-Cys-Gly,is the
redox buffer in cells,cycling between GSH
(reduced form) and GSSG (oxidized form);
? The tripeptide is synthesized using specific
enzymes (not ribosomes)
The plant growth hormone,
indole-3-acetate,is derived
from Trp.
Many neurotransmitters are
derived from amino acids
Spermidine and spermine
are derived from Met
and ornithine.
Nitric oxide (NO) is made from Arg and O2 in a reaction
catalyzed by nitric oxide synthase (NOS),a
monooxygenase containing five coenzymes,NADP,FMN,
FAD,tetrahydrobiopterin,and a Fe 3+-heme,
(Robert F,Furchgott,Louis J,Ignarro,and Ferid Murad were
awarded the 1998 Nobel Prize in Medicine for revealing the
role of NO in the cardiovascular systems)
18,Nucleotides are synthesized via
either the de novo pathways or the
salvage pathways
? In the de novo pathway simple precursors,
including amino acids,PRPP,NH4+,CO2,
and one-carbon units (carried on H4
folate) are used.
? In the salvage pathway the free bases and
nucleosides released from nucleic acid
breakdown are used.
? The free bases (A,G,C,U,T) are not intermediates
during the de novo synthesis,the purine ring is
assembled on ribose phosphate to make AMP and
GMP; the pyrimidine ring is first synthesized as
orotate,which is then attached to ribose phosphate
before being converted to UTP and CTP (dTMP is
made from dUMP).
? The deoxyribonucleotides (dNDPs) are synthesized
by reduction of ribonucleotides (NDPs).
19,Radioisotope tracer experiments
revealed the origins of the atoms in
the purine and pyrimidine rings
? Buchanan and Greenberg revealed this by feeding a
variety of isotopically labeled compounds to pigeons
(1940s).
? The atoms of the purine rings were found to be
derived from formate,CO2,Gly,Asp,and Gln.
? The atoms of the pyrimidine rings were found to be
derived from Asp,Gln and HCO3-.
Radioisotope tracer experiments revealed
the origins of the ring atoms of purines
Gln amide
HCO3-
C
The atoms of the pyrimidine rings were revealed to
Be derived from HCO3-,Gln and Asp.
20,De novo purine nucleotide
synthesis begins with the transferring
of an amino group from Gln to PRPP
? PRPP is synthesized from ribose 5-P.
? In the first (committing) step of purine nucleotide synthesis,
the PPi part on PRPP (at C-1) is replaced by the side chain
amino group of Gln,forming 5-phosphoribosylamine.
? A glycine (2C +1N),a N10-formyl H4 folate (1C),a Gln
(1N),a HCO3-(1C) Asp (1N),and another N10-formyl H4
folate (1C) then brings the rest atoms for the purine ring.
? The first intermediate having a complete purine ring
is IMP (inosinate).
? IMP is converted to AMP by accepting an amino
group from Asp,and converted to GMP by a
NAD+-dependent dehydrogenation reaction (at C-2)
and an amino group transfer from Gln (catalyzed by
an amidotransferase).
? The production of AMP from IMP requires GTP,
and the production of GMP from IMP requires ATP.
PRPP is synthesized from ribose 5-P in a
Reaction catalyzed by PRPP synthetase.
The purine ring is built up one or a few atoms at
a time on ribose 5-P
Steps 1,3,and 5 are
catalyzed by one
multifunctional protein
in some eukaryotic cells.
IMP is first
formed and
is then converted
to AMP and
GMP.
Steps 7 and 8 are
catalyzed by one
protein in some
eukaryotic cells
Steps 10 and 11 are
catalyzed by one protein
in some eukaryotic cells
IMP is converted to AMP by accepting an amino
group from Asp (GTP is needed to activate Asp) and to
GMP by accepting an amino group from Gln (ATP is
needed to activate XMP)
21,The biosynthesis of AMP and
GMP is regulated by feedback
inhibition
PRPP synthetase and glutamine-PRPP
amidotransferase are both inhibited by the end
products IMP,AMP and GMP (AMP and
GMP act synergistically).
AMP inhibits adenylosuccinate synthetase and
GMP inhibits IMP dehydrogenase.
AMP and GMP synthesis is balanced by the
following mechanism,GTP is needed for
AMP synthesis and ATP for GMP synthesis.
The de novo synthesis
of AMP and GMP
is regulated mainly
by sequential feedback
Inhibition.
22,The de novo biosynthesis of
pyrimidines begin with the formation
of carbamoyl phosphate
? Carbamoyl phosphate is formed from Gln,HCO3-,
and ATP in a reaction catalyzed by the carbamoyl
phosphate synthetase II.
? In the committing step of pyrimidine biosynthesis,
carbamoyl phosphate condenses with Asp to form
N-carbamoylaspartate in a reaction catalyzed by
aspartate transcarbamoylase (ATCase).
? A ring closing reaction generates L-dihydroorotate,
which is then oxidized to form orotate (乳清酸 ) in a
reaction catalyzed by dihydroorotate dehydrogenase.
? Orotate is then linked to PRPP to form orotidylate,
which is then converted to UMP by a decarboxylation
reaction.
? CTP is derived from UTP by accepting an amino
group from Gln or NH4+.
? In eukaryotic cells,carbamoyl phosphate synthetase
II,aspartate transcarbamoylase,dihydroorotase,and
are part of a trifunctional proteins called CAD.
? When PALA,an ATCase inhibitor was added
to cultured mammalian cells,concentrations of
all three enzymes are increased dramatically
for cells resistant to PALA.
Gln
HCO3-+ATP
NH4+
Carbamoyl phosphate
Intermediates in
bacterial carbamoyl
phosphate synthetase
is channeled.
23,Pyrimidine nucleotide biosynthesis
is regulated at the aspartate
transcarbamoylase (ATCase)
? ATCase is inhibited by CTP,the end product of the
de novo pyrimidine nucleotide biosynthesis pathway.
? CTP binds at the regulatory subunits,which changes
the conformation of the catalytic subunits,
inactivating them.
? ATP is able to prevent the changes induced by CTP.
ATCase contains separate catalytic and
regulatory subunits
Catalytic
Subunits (binding Asp and carbamoyl phosphate)
Regulatory subunits (binding CTP or ATP)
24,Base specific NMP kinases
together with a nonspecific NDP
kinase converts NMPs to NTPs
? Nucleotides participate in biosynthesis (of RNA and
DNA) in the forms of NTPs.
? Each specific nucleoside monophosphate (NMP)
kinase converts the corresponding NMP/dNMPs to
NDP/dNDPs using ATP.
? The nonspecific nucleoside diphosphate (NDP)
kinase converts all NDP/dNDPs to NTP/dNTPs
using ATP or other NTPs.
25,Deoxyribonucleotides are made
from ribonucleotides at the NDP
level
? This occurs by direct reduction at the 2`-carbon.
? Ribonucleotide reductase catalyzes all such
conversions.
? The electrons are provided by NADPH and is
transferred to the ribonucleotide reductase via two
possible paths (via either thioredoxin or
glutaredoxin).
NDPs are converted
to dNDPs with the
catalysis of
ribonucleotide
reductase,with
electrons ultimately
coming from NADPH
Electrons are transferred from NADPH to the ribonucleotides
Via thioredoxin
(硫氧还蛋白 ）
Via glutaredoxin
(谷氧还蛋白 ）
26,Free radicals are involved in
converting NDPs to dNDPs by the
ribonucleotide reductase
? Ribonucleotide reductase exists as a tetramer,with
two R1 and two R2 subunits.
? Each tetramer has two active sites formed at the
interface of R1 and R2 subunits,R1 contributes two –
SH groups and R2 contribute a radical (stabilized by
a Tyr radical,which,in turn is stabilized by a Fe3+-
Fe3+ binuclear center in class I ribonucleotide
reductase),
? The deoxygenation reaction is proposed to
occur via a 3`-ribonucleotide radical,which
help stabilize the 2` carbon cation
subsequently formed.
Ribonucleotide
reductase is a
tetramer of two
different subunits
The R2 dimer of
ribonucleotide reductase,
with a Tyr radical and a
Fe3+-Fe3+ binuclear center.
Tyr radical
Fe3+-Fe3+
A proposed
mechanism for
converting a
NDP to a dNDP
by ribonucleotide
reductase,the
radical property
of the enzyme is
transiently
transferred to the
substrate; the
presence of the
radical at C-3
stabilizes the
carbon cation
formed at C-2.
27,The E,coli ribonucleotide
reductase is regulated for both its
activity and substrate specificity
? Two types of regulatory sites are present on the R1
subunits,one for substrate specificity and the other
for overall enzyme activity.
? At the substrate specificity site,when dATP or ATP
binds,reduction of CDP and UDP is favored; when
dTTP binds,reduction of GDP is favored (and the
reduction of CDP and UDP is inhibited); when
dGTP binds,reduction of ADP is favored.
? At the overall enzymatic activity site,when
ATP binds,the enzyme is activated; when
dATP binds,the enzyme is inactivated.
Both the substrate specificity and
the overall enzymatic activity of
ribonucleotide reductase is
regulated to balance the
biosynthesis of all nucleotides.
28,dTMP is synthesized by
methylation of dUMP
? dUTP is first formed from either dUDP (via
phosphorylation) or dCTP (via deamination).
? dUMP is then formed from dUTP in a reaction
catalyzed by dUTPase (keeping dUTP at a low level
to prevent its incorporation into DNA).
? dUMP is then converted to dTMP by the catalysis of
thymidylate synthase,with a methylene group
transferred from and reduced by N5,N10-methylene
H4 folate (being donors of both one-carbon unit and
electrons!).
? The dihydrofolate is reduced to H4 folate by
NADPH in a reaction catalyzed by dihydrofolate
reductase (DHFR).
? The N5,N10-methylene H4 folate is regenerated from
Ser and tetrahydrofolate in a reaction catalyzed by
serine hydroxymethyltransferase.
? The reactions catalyzed by ribonucleotide
reductase and thymidylate synthase are probably
key for the transition from an RNA world to one in
which DNA stores genetic information.
dTMP is derived from dUMP via a methylation
reaction using N5,N10-methylene H4 folate as
donors of both one-carbon unit and electrons.
dUMP is converted to
dTMP in a N5,N10-
methylene H4 folate-
dependent reaction
catalyzed by
thymidylate synthase.
29,Uric acid is the excreted end
product of purine catabolism in
humans and many other animals
? For adenosine,the amino group is hydrolyzed before
the ribose group is removed.
? For guanosine,the amino group is hydrolyzed after
the ribose group is removed.
? Xanthine oxidase,having multiple cofactors
(including an FAD,a Mo complex,four different
Fe-S clusters),catalyzes the O2-dependent
conversion of hypoxanthine to xanthine and
xanthine to uric acid.
? Uric acid can be further converted to allantoin,
allantoate,urea or NH4+ in various animals.
? The deficiency of adenosine deaminase causes the
severe immunodeficiency disease in humans (it is
likely the accumulated adenosine is converted to
dATP,which inhibits the formation of all dNDPs by
ribonucleotide reductase).
? Overproduction of uric acid was revealed to cause
gout (痛风 ).
? Allopurinol,an inhibitor of xanthine oxidase,is used
to treat gout.
(黄嘌呤 )
(尿酸 )
(尿囊素 ）
(尿囊酸 )
Dephosphorylation
Dephosphorylation
Deamination
Deamination
DeribosylationDeribosylation
Oxidation
Oxidation
Allopurinol was designed to be a competitive inhibitor of
xanthine oxidase to treat gout by Elion and Hitchings,who
shared the Nobel Prize in 1988 for their discoveries of
important principles for drug treatment
30,Pyrimidines are broken down via
reduction
? The degradation of thymine produces
mathylmalonyl-CoA,which can be converted to
succinyl-CoA (a citric acid cycle intermediate) by
the catalysis of a mutase.
? The degradation of uracil and cytidine produces
malonyl-CoA,which is one precursor for fatty acid
biosynthesis.
? To a limited extent,catabolism of pyrimidine
nucleotides contributes to the energy metabolism of
the cell.
Propionyl-CoA Succinyl-CoA
Degradation products of
pyrimdines can enter the
citric acid cycle
31,Purine and pyrimidine bases can
be reconverted into nucleotides via
the salvage pathway
? Adenine phosphoribosyltransferase catalyzes the synthesis
of AMP from adenine and PRPP.
? Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
catalyzes the synthesis of GMP and IMP.
? The lack of HGPRT will cause Lesch-Nyhan syndrome.
? Pyrimidine bases are recycled in a similar way in
microorganisms,but pyrimidine bases does not seem to be
salvaged in significant amounts in mammals.
The purine bases can be converted
to purine nucleotides via the
salvage pathways
32,Many cancer chemotherapeutic
drugs target enzymes in the
nucleotide biosynthetic pathways
? Analogs of Gln,like azaserine and acivicin,inhibits many
amidotransferases used in nucleotide (and amino acid)
biosynthesis.
? Fluorouracil,after being converted to FdUMP by the
salvage pathway,can inhibit the thymidylate synthase after
two steps of conversion,thus inhibit the dTMP synthesis.
? Methotrexate,a folate analog,inhibits the dihydrofolate
reductase,thus the dTMP synthesis.
Azaserine and acivicin
inhibits amidotransferases,
thus inhibit the biosynthesis
of nucleotides and amino
acids.
FdUMP (from fluorouracil)
and methotrexate inhibits
thymidylate synthase and
DHFR respectively.
FdUMP is an suicide inhibitor of
thymidylate synthase
Summary
? Atmospheric N2 is reduced to ammonia by
the dinitrogenase reductase and the
dinitrogenase (containing a key Fe-Mo
cofactor) of the nitrogenase complex present
only in certain bacteria.
? Ammonia enters organic molecules via Glu
and Gln.
? Glutamine amidotransferases catalyzes the
transferring of the amide amino group to
many acceptor molecules.
? Amino acids are mainly derived from intermediates
of glycolysis,the citric acid cycle,and the pentose
phosphate pathway.
? Pro and Arg are derived from Asp,which is
synthesized from a-ketoglutarate.
? Ser,Gly,and Cys are derived from 3-
phosphoglycerate.
? Lys is derived from oxaloacetate (as Asp),pyruvate
and Glu; Met is derived from oxaloacetate (as Asp),
Cys,and N5-methyl H4 folate; Thr is derived from
oxaloacetate.
? Ile and Val are derived from Thr/pyruvate and two
molecules of pyruvate respectively,using the same
enzymes; Leu is derived from two molecules of
pyruvate,sharing four steps of reactions with Val
synthesis.
? Trptophan is derived from part of the atoms of two
molecules of phosphoenolpyruvate,one erythrose 4-
P,one GLn,one PRPP,and one Ser; Phe and Tyr
are synthesized from two phosphoenolpyruvates,
one erythrose 4-P,and one Glu.
? His is derived from one PRPP,one ATP,One Gln,
and one Glu.
? Amino acid biosynthesis is regulated by various
forms of feedback inhibition (including enzyme
multiplicity,concerted inhibition and sequential
feedback inhibition).
? Many other biomolecules (including hemes,creatine,
glutathione,plant growth hormones,
neurotransmitters,polyamies,and nitric oxide) are
derived from amino acids.
? Purine nucleotides are synthesized from PRPP,Gln,
Gly,N10-formyl H4 folate,Gln,HCO3-,Asp through
the de novo pathway.
? Pyrimidine nucleotides are synthesized using HCO3-,
Gln,Asp,and PRPP.
? De novo synthesis of nucleotides are regulated via
feedback inhibition (no covalent modifications yet
revealed).
? Deoxyribonucleotides are derived from
ribonucleotides at the NDP level,with the catalysis
of ribonucleotide reductase,which contains a chain
of electron carriers,uses free radicals,and being
regulated for both substrate specificity and overall
enzymatic activities.
? The dTMP molecule is derived from dUMP by
thymidylate synthase,an enzyme using N5,N10-
methylene-tetrahydrofolate as the donor of both one-
carbon unit and electrons.
? Degradation of purines and pyrimidines produces uric
acid and citric acid cycle intermediate/fatty acid
synthesis precursor,respectively.
? Purine and pyrimidine bases can be reused via the
salvage pathway.
? Many cancer chemotherapeutic drugs (e.g.,azaserine,
acivicin,fluorouracil,and methotrexate) inhibits
enzymes in the nucleotide biosynthetic pathways.
