Amino Acid Metabolism 1: Nitrogen fixation and assimilation, amino acid degradation, the urea cycle

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Amino Acid Metabolism 1Nitrogen fixation and
assimilation, amino acid degradation, the urea
cycle
Bioc 460 Spring 2008 - Lecture 38 (Miesfeld)
Glutamine synthetase converts glutamate to
glutamine through a nitrogen assimilation reaction
Urea is a nitrogen-containing metabolite that
efficiently removes toxic ammonia
Red clover is a leguminous plant that is often
used in crop rotation strategies
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Key Concepts in Amino Acid Metabolism

• Certain types of bacteria can use nitrogen
  fixation reactions to convert atmospheric N2 into
  NH4.
• The enzymes glutamate synthase, glutamine
  synthetase, glutamate dehydrogenase, and
  aminotransferases are responsible for the vast
  majority of nitrogen metabolizing reactions in
  most organisms.
• Protein degradation by the proteasomal complex
  releases oligopeptides that are degraded into
  individual amino acids.
• The urea cycle uses nitrogen from NH4 and the
  amino acid aspartate to generate urea which is
  excreted to maintain daily nitrogen balance.

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Amino Acid Metabolism
The carbon skeleton of amino acids can be
harvested for energy converting reactions,
whereas, the nitrogen is safely removed to avoid
ammonia toxicity.
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Nitrogen fixation and assimilation by plants and
bacteria
1. What purpose does nitrogen fixation and
assimilation serve in the biosphere? Nitrogen
fixation takes place in bacteria and is the
primary process by which atmospheric N2 gas is
converted to ammonia (NH4) and nitrogen oxides
(NO2- and NO3-) in the biosphere. Nitrogen
assimilation incorporates this ammonia into amino
acids, primarily glutamate and glutamine. 2.
What are the net reactions of nitrogen fixation
and assimilation by plants and bacteria? Nitrogen
fixation is mediated by the nitrogenase enzyme
complex N2 8 H 8 e- 16 ATP 16 H2O
----gt 2 NH3 H2 16 ADP 16 Pi Nitrogen
assimilation using glutamine synthetase and
glutamate synthase a-ketoglutarate NH4
ATP NADPH H --gt Glutamate ADP Pi
NADP
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Nitrogen fixation and assimilation by plants and
bacteria
3. What are the key enzymes in nitrogen fixation
and assimilation? Bacterial nitrogenase complex
is the enzyme that uses redox reactions coupled
to ATP hydrolysis to convert N2 gas into 2 NH3.
Glutamine synthetase - is found in all
organisms and it incorporates NH4 into glutamate
to form glutamine through an ATP coupled redox
reaction. Glutamate synthase - is found in
bacteria, plants, and some insects, and it works
in concert with glutamine synthetase to replenish
glutamate so that the glutamine synthetase
reaction is not substrate limited. Glutamate
dehydrogenase - is found in all organisms and it
interconverts glutamate, NH4, and
a-ketoglutarate in a redox reaction utilizing
either NAD(P)/NAD(P)H.
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Nitrogen fixation and assimilation by plants and
bacteria
4. What are examples of nitrogen fixation and
assimilation in real life? Natural fertilizers
can be used in organic farming to reduce the
dependence on industrial sources of nitrogen. By
plowing under the leguminous plants, the nitrogen
contained in the plants is released into the soil
and processed by soil bacteria to provide
nitrogenous compounds for corn and wheat plants.
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Nitrogen fixation
Rhizobium bacterium
Tucson lightning
The Haber process
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Nitrogen fixation in bacteria
Biological nitrogen fixation by bacteria requires
the activity of nitrogenase, a large protein
complex consisting of two functional components.
One component is called dinitrogenase reductase
(Fe-protein) which consists of two identical
subunits that each contain a binding site for
ATP, and a single 4Fe-4S redox center liganded to
cysteine residues in the two subunits.
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Nitrogenase reaction is a series of reductions
N2 8 H 8 e- 16 ATP 16 H2O ----gt 2 NH3
H2 16 ADP 16 Pi
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Nitrogen fixation in bacteria
Rhizobium meiloti is one of the bacterial species
that is capable of nitrogen fixation. This
bacterial species invades the roots of leguminous
plants through tubular structures called
infection threads.
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Nitrogen assimilation in plants
Nitrogen assimilation proceeds in one of two
ways. First, if NH4 levels in the soil are
high, plants can use the glutamate dehydrogenase
reaction to directly incorporate NH4 into the
amino acid glutamate using a-ketoglutarate as the
carbon skeleton.
Note that most often, this reaction releases NH4
from glutamate in other contexts.
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Nitrogen assimilation in plants
A second, and more common way that plants and
bacteria incorporate NH4 into metabolites, is
through a two reaction mechanism that functions
when NH4 concentrations are low. In this
mechanism, the enzyme glutamine synthetase uses
ATP in a coupled reaction to form glutamine from
glutamate using NH4.
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Nitrogen assimilation in plants
Next, the glutamine is combined with
a-ketoglutarate in a reaction catalyzed by the
enzyme glutamate synthase to form two molecules
of glutamate (glutamine contains two nitrogens).
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The net reaction is nitrogen assimilation
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Importantly, the newly acquired nitrogen in
glutamate and glutamine is used to synthesize a
variety of other amino acids through
aminotransferase enzymes such as aspartate
aminotransferase.
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The Nitrogen Cycle on Planet Earth
The Nitrogen Cycle maintains nitrogen balance in
our biosphere. The NH4 in the soil derived
from decomposition, free-living soil bacteria,
and man-made fertilizers, is converted to NO2-
(nitrite) and NO3- (nitrate) by soil bacteria
that carry out the process of nitrification
(e.g., Nitrosomonas and Nitrobacter). Plant
roots absorb NO2- and NO3- present in the soil
and convert them back into NH4 using nitrite and
nitrate reductase enzymes.
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Protein and amino acid degradation
A normal healthy adult needs about 400 grams of
protein per day to maintain nitrogen balance.
In contrast, young children and pregnant women
have a positive nitrogen balance because they
accumulate nitrogen in the form of new protein
which is needed to support tissue growth.
Negative nitrogen balance is a sign of disease
or starvation and occurs in individuals with
elevated rates of protein breakdown (loss of
muscle tissue) or an inability to obtain
sufficient amounts of amino acids in their diet.

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Protein digestion in humans takes place in the
stomach and the small intestine where proteases
cleave the peptide bond to yield amino acids and
small oligopeptides.
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Protein and amino acid degradation
The duodenum secretes enteropeptidase, a protease
that specifically activates several protealytic
zymogens released from the pancreas. One of
these proteases is the pancreatic zymogen
trypsinogen which is cleaved to form the
endopeptidase trypsin that cleaves numerous other
pancreatic zymogens.
Trypsin autoactivates
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Protein and amino acid degradation

• Most eukaryotic cellular proteins are degraded by
  one of two pathways
• ATP-independent process that degrades proteins
  inside cellular vesicles called lysosomes.
• ATP-dependent pathway that targets specific
  proteins for degradation in proteasomes if they
  contain a polymer of ubiquitin protein covalently
  attached to lysine residues.

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E1 passes it to E2
E1 is ubiquinated
E2 E3 ubiquinate the target protein
Ubiquitin is the molecular signal for degradation
Ubiquitin is recycled and the protein is degraded
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The 26S proteasome consists of a 20S protealytic
core and two 19S regulatory complexes that serve
as caps to regulate protein entry.
The "garbage disposer" of the cell
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Amino acids are stripped of their carbon
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The Urea Cycle
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1. What does the urea cycle accomplish for the
organism? Urea synthesis provides an efficient
mechanism for land animals to remove excess
nitrogen from the body. Urea is synthesized in
the liver and exported to the kidneys where it
enters the bladder. 2. What is the net reaction
of the urea cycle? NH4 HCO3- aspartate 3
ATP ---gt urea fumarate 2 ADP 2 Pi AMP
PPi 3. What is the key regulated enzyme in
urea synthesis? Carbamoyl phosphate synthetase I
catalyzes the commitment step in the urea
cycle the activity of this mitochondrial enzyme
is activated by N-acetylglutamate in response to
elevated levels of glutamate and arginine. 4.
What is an example of the urea cycle in real
life? Argininosuccinase deficiency inhibits flux
through the urea cycle and causes hyperammonemia
and neurological symptoms. This metabolic
disease can be treated with a low protein diet
that is supplemented with arginine, thereby
resulting in argininosuccinate excretion a
substitute for urea.
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Glutamate is imported into the mitochondrial
matrix where it is metabolized by the enzyme
glutamate dehydrogenase to produce NH4 which is
used to make the urea cycle precursor carbamoyl
phosphate.
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The Urea Cycle
By including the pyrophosphatase reaction
(argininosuccinate synthetase reaction), it can
be seen that four high energy phosphate bonds are
required (4 ATP equivalents) for every molecule
of urea that is synthesized, and moreover, that
the C4 carbon backbone of aspartate gives rise to
fumarate NH4 CO2 aspartate 3 ATP
---gt urea fumarate 2 ADP AMP 4
Pi
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The aspartate-argininosuccinate shunt converts
fumarate, produced in the cytosol by the urea
cycle, into malate that is used to make
oxaloacetate in the citrate cycle, thereby,
forming the Krebs bicycle.
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Enzyme Deficiencies in the Urea Cycle
Since argininosuccinate is soluble and can be
excreted in the urine, it functions as a
metabolic replacement for urea. Supplementing the
diet with ornithine would give the same result,
but it is more feasible to use arginine.
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Degradation of glucogenic and ketogenic amino
acids
The carbon backbones of eleven of the twenty
standard amino acids can be converted into
pyruvate or acetyl-CoA, which can then be used
for energy conversion by the citrate cycle and
oxidative phosphorylation reactions. The other
nine amino acids are converted to the citrate
cycle intermediates ?-ketoglutarate, fumarate,
succinyl-CoA, and oxaloacetate, which can be used
for glucose synthesis by conversion of
oxaloacetate to phosphoenolypyruvate. Under
normal conditions, amino acid degradation
accounts for 10-15 of the metabolic fuel for
animals, more so for animals with high protein
diets or during starvation when muscle protein is
degraded.
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Glucogenic and ketogenic amino acids