Isoleucine
Aldol cleavage
The a and b subunits of tryptophan
synthase have different enzymatic
activities, substrate channeling
occurs in this enzyme.
Dehydration of Ser
Substrate
channeling
Enzymes involved in Trp synthesis
may exist as multi-enzyme complexes
(metabolons).
Amino acids are mainly derived from
intermediates of glycolysis, the citric
acid cycle, and the pentose
phosphate pathway.
Pyruvate
The rate of these biosynthetic
pathways are often regulated
via allosteric feedback
inhibition.
The first case of allosteric
feedback inhibition was discovered
in studying the biosynthesis of
Ile in E. coli.
Umbarger, H. E. (1956) “Evidence for a negativefeedback mechanism in the biosynthesis of
isoleucine”, Science 123, 848.
Allosteric feedback inhibition for
Ile biosynthesis
The first catalytic enzyme
Ile inhibits this enzyme in
a minute-to-minute response.
End product
Concerted inhibition
Three aspartokinase isozymes
Enzyme multiplicity
Sequential
feedback
inhibition
Goal of regulation:
provide a balanced
supply of all amino
acids!
Interlocking regulatory
mechanisms in the
biosynthesis of Lys,
Met, Thr, and Ile from
Asp.
Amino acids are
precursors of many
specialized biomolecules
Glutathione
Melatonin
Catecholamine
neurotransmitters
like dopa, dopamine,
norpinephrine, and
eipnephrine are
derived from Tyr.
Nucleotide
biosynthesis
The eight nucleotides found in DNA and RNA
Issues: Sources of carbon and nitrogen; assembly order; balanced
synthesis of all the nucleotides; cofactors.
Deoxyribonucleotides are derived from ribonucleotides,
dTMP is derived from dUMP, supporting the “RNA world”
hypothesis of evolution.
Ribonucleotides, unlike deoxyribonucleotides, have multiple metabolic roles.
Nucleotides are
synthesized via
either the de
novo or salvage
pathways.
Radioisotope tracer experiments
(using 14C and 15N-labeled
precursors) in birds revealed the
origins, not the assembly pathway,
of the atoms in the purine and
pyrimidine rings (Buchanan and
Greenberg, 1950s).
Assembled onto a
ribose 5-P.
Origins of the ring
atoms of purines
Could the assembly
occur without
enzyme catalysis
(when enzymes were
not yet available)?
The ring (as orotate) is assembled
first before attaching to a ribose 5-P.
Gln amide
HCO3The atoms of the pyrimidine rings were mostly
derived from amino acids.
De novo purine nucleotide
synthesis: the base
assembles on the ribose
phosphate; IMP is the first
nucleotide synthesized.
Stepwise assembly of the purine
ring occurs on ribose phosphate
Ribose 5-P
Committing
step
(Unstable)
Nonsequential
steps 1, 3, 5 are
catalyzed by one
multifunctional
protein in
eukaryotes!
1st ring closure
(remnant of ATP released
during His biosynthesis)
Steps 10 and 11
are catalyzed by
one protein.
2nd ring
closure
Asp donates an
amino group in a
similar fashion
as it does in the
urea cycle.
Nucleotide biosynthesis occurs
via either the de novo or
salvage pathways
• For the de novo pathway, amino acids (Asp, Gln,
Gly), ribose 5-phosphate, CO2, One-carbon units
(carried by H4 folate) are the precursors.
• The free bases are not intermediates for the de
novo pathways.
• The purine ring is assembled on ribose 5-P,but the
pyrimidine ring is assembled first before attached
to ribose 5-P.
• Cellular pools of nucleotides are quite small
(continuous biosynthesis is needed).
• Preformed bases are recovered and reconnected to
a ribose unit in the salvage pathways.
GTP as an energy source!
Balanced synthesis
of AMP and GMP:
AMP formation
requires GTP &
GMP formation
requires ATP!
Biosynthesis of
AMP & GMP from IMP.
The biosynthesis of AMP
and GMP is regulated
mainly by sequential
feedback inhibition (no
covalent regulation)
AMP and GMP act synergistically.
Syntheses of
AMP & GMP
are balanced
via feedback
inhibition.
GTP
The committing step
+
+
ATP
The de novo biosynthesis of
pyrimidine nucleotides: the
base ring (as orotate) is
first assembled before
being attached to the
phosphoribosyl group to
form orotidylate.
Gln
A substrate channel
is found in carbamoyl
phosphate synthetase II
NH4+
HCO3- ( ATP)
ADP
Pyrimidine nucleotide
biosynthesis in bacteria is
regulated at the aspartate
transcarbamoylase (ATCase):
feedback inhibition by CTP.
CTP
CTP
CTP
Inhibited state
ATP
ATP
Active state
CTP
Feedback inhibition
of CTP
ATP
ATP
Bacterial ATCase,
one of the earliest
& best studied
allosteric enzymes,
contains separate
catalytic &
regulatory
subunits.
Base specific NMP kinases
together with a nonspecific NDP
kinase converts NMPs to NTPs
• Each base-specific nucleoside
monophosphate (NMP) kinase converts the
corresponding NMP/dNMPs to NDP/dNDPs
using ATP.
• The nonspecific (ubiquitous) nucleoside
diphosphate (NDP) kinase converts all
NDP/dNDPs to NTP/dNTPs using ATP or
other NTPs
Metal ions (Mg2+ or Mn2+) are found to be essential for these enzymes
to be active. ATP induced conformational changes prevents hydrolysis
All dNDPs are derived from
NDPs via the catalysis of a
common ribonucleotide
reductase likely via a 3`ribonucleotide radical
intermediate.
This is consistent with the notion that RNA
preceded DNA in the course of evolution!
Ribonucleotide
reductase
contains two
types of
subunits.
Shown is the E. coli
enzyme, based on
determined structure
of the R1 dimer and
R2 dimer (not as a
whole!)
A unique feature of
this enzyme
The action
mechanism
of this enzyme is
far from clear.
Radical at 3`-C
No activation
at C-2’!
Reverse of step 1
Proposed catalytic
mechanism for
ribonucleotide
reductase.
Loss of water
from C’-2
Radical cation
dTMP is synthesized by
the methylation of dUMP.
donors of one-carbon
units & electrons
Thymidylate synthase
and dihydrofolate
reductase reside in
one protein in plants.
dUMP is derived from dUTP, which is in turn derived
from dCTP or dUDP.
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.
Purine and pyrimidine bases
can be reconverted to
nucleotides via the salvage
pathway.
Salvage pathway of
nucleotide biosynthesis
Defect of HGPRT
causes Lysch-Nyhan
syndrome.
Adenine or hypoxanthine-guanine
phosphoribosyltransferases
Uric acid is the excreted
end product of purine
nucleotide catabolism in
humans and many other
animals.
AMP deminase
nucleotidase
Nucleotide
Oxidation of GMP
and AMP to produce
uric acid
nucleotidase
Overproduction of
uric acid, which
will accumulate
in the joints, was
revealed to cause
gout (痛风).
Nucleoside
Adenosine
Deaminase
ADA)
purine nucleoside
phosphorylase
Base
Nucleoside
guanine
deaminase
purine nucleoside
phosphorylase
The deficiency of
ADA will cause
severe combined Base
immunodefficiency
(SCID) in human.
Xanthine
oxidase
Xanthine
oxidase
Allopurinol was designed to be a suicide inhibitor of
xanthine oxidase to treat gout (Elion and Hitchings
shared the Nobel Prize in 1988 for their discoveries of
important principles for such drug design).
Uric acid is further degraded in many animals.
Amidotransferases,
thymidylate synthase and
dihydrofolate reductase
are target enzymes for
cancer chemotherapeutic
drugs (to inhibit the
synthesis of dTMP)
Analogs of Gln inhibit
synthesis of nucleotides and
some amino acids
Suicide inhibitor
of thymidylate
synthase
Competitive inhibitor of DHFR
Summary
• Atmospheric N2 is reduced to ammonia by
the the nitrogenase complex (containing a
key Fe-Mo cofactor) present only in
certain bacteria.
• Ammonia enters organic molecules via
Gln and Glu.
• Glutamine amidotransferases catalyzes
the transferring of the amide amino group
of Gln to many acceptor molecules.
• The carbon skeletons of amino acids are
mainly derived from intermediates of
glycolysis, the citric acid cycle, and the
pentose phosphate pathway.
• Amino acid biosynthesis is regulated by
various forms of feedback inhibition
(including enzyme multiplicity, concerted
inhibition and sequential feedback
inhibition).
• Many other biomolecules are derived
from amino acids.
• Purine nucleotides are synthesized
from PRPP, Gln, Gly, N10-formyl H4
folate, 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.
• 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 be regulated for
both substrate specificity and overall
enzymatic activities.
• The dTMP molecule is derived from
dUMP by thymidylate synthase, an
enzyme using N5, N10-methylenetetrahydrofolate 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.