Transcript Chapter 21 Biosynthesis of amino acids, nucleotides and related

For Biochemistry II, Dec. 16 and 31, 2009
Chapter 22
Biosynthesis of amino acids,
nucleotide
To be lectured by Professor Zengyi Chang
1.
2.
3.
4.
Source of nitrogen.
Source of carbon.
De novo and salvage pathways.
Ways to balance the synthesis of each.
Overview
The 20 standard amino acids are usually categorized into five families
Issues: What are used as precursors to generate the carbon skeletons
what are the chemical processes and enzymes involved
How are the processes related to each other
Why only L-amine acids are synthesized in the cells
How would a balanced synthesis of each amino acid be achieved
Tyrosine
cysteine
The eight nucleotides found in DNA and RNA
Issues: How are the base, the sugar, & the phosphate assembled
What are the starting precursors
What chemical processes and enzymes are involved
Are the synthetic processes related to each other
How would a balanced synthesis of each to be achieved
The biosyntheses of the 20 amino
acids can be grouped into six families
Nutritional requirements for
amino acids in mammals
( the a-keto acid not synthesized!)
Amino acids also function as
precursors to hormones,
coenzymes, porphyrins, pigments,
Neurotransmitters, etc.
Nutritional quality of proteins for humans:
Mammals
> fish & poultry > fruits & plants.
The biosynthesis of nucleotides:
an outline
Gln +
HCO3-
The purine ring
is assembled
on ribose
phosphate.
NH2
C
O
O
PO3-
The pyrimidine
ring is assembled
first before attached
to Ribose phosphate.
Difficulties and importance
of studying this chapter
• Many pathways involve many steps and
intermediates.
• Some most unusual chemical
transformations in biosystems found here.
• Many genetic diseases are caused by
defects of enzymes discussed here.
• Many pharmaceuticals in common use to
combat infectious diseases or cancer are
inhibitors of enzymes discussed here.
• Best-understood examples of enzyme
regulation are seen here.
Most organisms maintain strict
economy in their use of
ammonia, amino acids and
nucleotides
• Biologically useful nitrogen
compounds are generally scarce in
the natural environments.
• Free amino acids, pyrimidines and
purines formed from metabolic
turnover are often salvaged (reused).
• Only certain bacteria are able to fix N2
into ammonia (NH3 or NH4+).
Biosynthesis of Amino acids
and nucleotides are closely
related
• Nitrogen arises from common biological
sources (N2 fixation).
• The two sets of pathways are extensively
intertwined (shared intermediates).
• Much common chemistry are found in
both pathways: transfer of nitrogen (often
from Gln) or one-carbon units (carried on
tetrahydrofolate).
• From where does nitrogen come from
Relationships between
Inorganic and organic
nitrogen metabolism
Few organisms can use the
N2 in air, and many soils are
poor in nitrate:
Nitrogen bioavailability limits
growth for most organisms
(thus the world’s food supply)!
Nitrogen (azote) enters biomolecules
via amino acids (revealed by using
radio isotopes, 15N and 14C)
Certain
bacteria
N2 → ammonia → Gln/Glu
→other
biomolecules
Highly comparable with CO2 fixation:
CO2 → 3-phospohglycerate → hexose
→ other biomolecules.
Both are highly energy consuming, needing NADPH and ATP!
N2 fixation is
thermodynamically favorable,
kinetically extremely slow
Has a bond energy of 930 kJ/mol
(while that for a C-O is 350 kJ/mol)
Biological N2 fixation in diazotrophs:
N2+8H++8e−+16ATP → 2NH3+H2+16ADP+16Pi
Here ATP hydrolysis reduces the heights of the
activation energy barrier, instead of for
thermodynamical purposes. The precise number of
ATP consumed in this process has not yet been established.
Cyanobacteria
Rhizobia
Biological nitrogen fixation
was first discovered
by Martinus Beijerinck,
a Dutch microbiologist
(1886).
Nitrogen fixation is catalyzed
by the nitrogenase complex,
present only in certain bacteria
(diazotrophs like cyanobacteria
and rhizobia) and energetically
costly.
The Haber method: N2 +3H2
2NH3 G`o = - 33.5kJ/mol
with iron catalyst, 500oC, 300 atm.
Can we design a process of producing ammonia under milder condition
by learning from what bacteria do in fixing nitrogen?
Nitrogenase reductase
(Fe Protein)
Electron
Donors
(ferredoxn or
Flavodoxin)
Nitrogenase (MoFe Protein)
8e− are needed to reduce each N2.
The
nitrogenase
complex
ATP binding and
hydrolysis
is thought to both
drive
the reduction of
the
P-cluster & to
trigger
a conformational
change in the
reductase
that causes it to
dissociate
transiently
from the
nitrogenase,
assuring
unidirectional
electron flow.
The nitrogenase complex is extremely
labile to O2 and various protective
mechanisms have evolved: living
anaerobically, forming thick walls,
uncoupling e- transport from ATP
synthesis (entering O2 is used
immediately) or being protected by
O2-binding proteins (e.g.,
leghemoglobin)..
Cyanobacteria
Rhizobia
heterocyst
Reduced nitrogen in the form
+
of NH4 is assimilated into
amino acids mainly via a
two-enzyme pathway :
glutamine synthetase and
glutamate synthase (an
enzyme only present in
bacteria and plants).
Ammonia enters organic
compounds in bacteria and
plants mainly via Gln and Glu
Gln synthetase
(present in all organisms)
Act to detoxify
ammonia
in animals!
Glu synthase
(present only in bacteria and plants)
The combined action of Gln synthetase and Glu synthase leads to the
net synthesis of Glu from a-ketoglutarate and NH4+!
The Glutamine synthetase is
a primary regulatory point
in nitrogen metabolism:
being regulated by at least
eight allosteric effectors
and reversible adenylylation
in prokaryotes.
The glutamine synthesis is constantly
tailored to cellular needs!
The E. coli glutamine synthetas
has 12 subunits (dodecamers)
arranged as two rings of hexamer
Active sites
at interfaces
Mn
Tyr397
(adenylylation site)
The glutamine
synthetase is
cumulatively
inhibited by at
least 8 allosteric
effectors, mostly
end products
of glutamine
metabolism.
Each of the 50 kDa
subunit contains
binding sites for
all the 8 allosteric
effectors in addition
to the active sites!
Gln
synthetase
AMP
The inactive form
Adenylylation increases the sensitivity
f each subunit to the 8 allosteric inhibitors.
Tyr397
A specific Tyr residue in bacterial glutamine synthetase
can be reversibly adenylylated by the catalysis of
adenylyltransferase (AT), whose activity is modulated by
a regulatory protein (PII), whose activity is in turn
regulated by uridylylation, catalyzed again by a single
The activity of E. coli Gln synthetase is regulated by reversible adenylylation.
Adenylyltransferase
Uridylyltransferase
Adenylyltransferase
Consequence of the regulation:
high Gln level
→ low Gln synthetase activity;
High a-ketoglutarate → high Gln synthetase activity.
“Activated
nitrogen”
The animal Gln synthetase seems to be regulated by changing
its oligomeric status (octameric to tetrameric).
Amidotransferases: a
family of enzymes that
catalyze the donation of
the amide amino group
from Gln to many other
“acceptor” compounds.
The biosythesis of 20 standard
amino acids
Highly conserved
Two-domain enzymes
Varies
A proposed general
action mechanism for
amidotransferases.
The carbon skeletons of
the 20 amino acids (in Lconfiguration) are derived
mainly from intermediates
of glycolysis, citric acid
cycle, and pentose
phosphate pathway in
bacteria and plants.
Pathways for synthesizing the
“essential” amino acids are usually
complex, involving 5-16 steps.
Pyridoxal phosophate and
tetrahydrofolate are two
cofactors widely used in
amino acid metabolism
Pyridoxal
phosophate
Tetrahydrofolate
(carries one-carbon units)