Download Lecture 38 - Amino Acid Metabolism 1

Amino Acid Metabolism 1:
Nitrogen fixation and assimilation, amino acid
degradation, the urea cycle
Bioc 460 Spring 2008 - Lecture 38 (Miesfeld)
Red clover is a leguminous
plant that is often used in
crop rotation strategies
Glutamine synthetase
converts glutamate to
glutamine through a nitrogen
assimilation reaction
Urea is a nitrogen-containing
metabolite that efficiently
removes toxic ammonia
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.
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.
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 ----> 2 NH3 + H2 + 16 ADP + 16 Pi
Nitrogen assimilation using glutamine synthetase and glutamate synthase:
a-ketoglutarate + NH4+ + ATP + NADPH + H+
--> Glutamate + ADP + Pi + NADP+
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.
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.
Nitrogen fixation
Rhizobium bacterium
Tucson lightning
The Haber process
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 4Fe4S redox center liganded to cysteine residues in the two subunits.
Nitrogenase reaction is a series of reductions
N2 + 8 H+ 8 e- + 16 ATP + 16 H2O ----> 2 NH3 + H2 + 16 ADP + 16 Pi
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.
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.
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+.
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).
The net reaction is nitrogen assimilation
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.
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.
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.
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.
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
Protein and amino acid degradation
Most eukaryotic cellular proteins are degraded by one of two pathways:
1) ATP-independent process that degrades proteins inside cellular
vesicles called lysosomes.
2) ATP-dependent pathway that targets specific proteins for
degradation in proteasomes if they contain a polymer of ubiquitin
protein covalently attached to lysine residues.
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
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
Amino acids are stripped of their carbon
The Urea Cycle
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 --->
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 Nacetylglutamate 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.
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.
Citrate
cycle
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 --->
urea + fumarate + 2 ADP + AMP + 4 Pi
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.”
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.
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 a-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 ~1015% of the metabolic fuel for animals, more so for animals with high
protein diets or during starvation when muscle protein is degraded.
Glucogenic
and ketogenic
amino acids