Nitrogen Acquisition and

Amino Acid Metabolism

Lesson Learning Outcomes

Upon completion of this lecture, students should be able to:

• explain amino acids role in nitrogen cycle
• explain nitrogen biosynthesis and catabolism of amino acids
• explain urea cycle
• The atmospheric nitrogen (N2) is not highly reactive
• but must be converted to ammonia (NH3) or nitrates (NO3-) to be biologically
useful
• Nitrogen fixation; requires electrons and energy

• 2 ways, lightning
• and nitrogen-fixing bacteria
• Symbotic organism that forms nodules on the roots of leguminous plants (soy beans,
peanuts, peas…)
• N2 to NH3 or more accurately the conjugate acid form of ammonia, the ammonium ion
(NH4+)
The nitrogen cycle. Organic nitrogenous compounds are formed by the
incorporation of NH4+ into carbon skeletons. Ammonium can be formed
from oxidized inorganic percursors by reductive reactions: nitrogen
fixation reduces N2 to NH4+; nitrate assimilation reduces NO3- to NH4+.
Nitrifying bacteria can oxidize NH4+ back to NO3- and obtain energy for
growth in the process of nitrification. Denitrification is a form of
bacterial respiration whereby nitrogen oxides serve as electron
acceptors in the place of O2 under anaerobic conditions.
• How N2 are metabolized in the ecology system?
• Nitrogen fixation
• Nitrification
• Denitrification
• Nitrification
biological oxidation of ammonia/ammonium to nitrite/nitrate
NO2 and NO3 is a nitrogen source for energy in organism growth

• Denitrification

• Reduction of nitrate ion NO3 and nitrite ion NO2- to molecular nitrogen (N2)
to the atmosphere
• The reduction of nitric oxide to nitrous oxide is mediated in nature by the NO
reductases (NOR), which occur in bacteria (NorBC) as well as in fungi
(P450nor)
 How nitrogen fixation happens?
 The nitrogenase enzyme complex in nitrogen-fixing bacteria catalyzes
the production of ammonia from molecular nitrogen
 Six (6) electrons are used to reduce molecular nitrogen to ammonium
ion
 Additional two (2) electrons are needed reduce hydrogen ions to H2
 Total reaction catalyzed by nitrogenase is an eight (8) electron reduction
 Several proteins are included in the nitrogenase complex – one of it is
ferredoxin
 Nitrogen fixing bacteria (roots of plants), free living microbes and
cyanobacteria are nitrogen fixing agents
Amino Acid Biosynthesis
• 10-15% total energy production
• Ammonia is toxic in high concentrations and must be incorporated
into biologically useful compounds
• Amino acid glutamate and glutamine are of central importance in the
process
• Glutamate is made from α-ketoglutarate, and glutamine is made
from glutamate
Amino acid synthesis
• NH4+ toxic!
• Incorporate into amino acids and nucleic acids
• Reductive amination using alpha-ketoglutarate

Glutamate dehydrogenase

• Amidation
Glutamine synthetase
 reductive amination amidation

• Production of glutamate is a reductive amination

• Production of glutamine is amidation
• Other reactions of amino acid anabolism, the alpha
amino group of glutamate and the side-chain
amino group of glutamine are shifted to other
compounds in transamination reactions
Transamination

• In transamination, the NH2 group on one molecule

is exchanged with the =O group on the other
molecule.
• The amino acid  keto acid & keto acid  amino
acid
• This process is an important step in the synthesis of
some non-essential amino acids (amino acids that
can be synthesized de novo by the organism)
• The primary mechanism for amino acid biosynthesis is via
the glutamate-dependent transamination of a-keto acid
carbon skeletons.
• The generic transamination - aminotransferase
reaction involves the transfer of the a-amino group of
glutamate to an a-keto acid acceptor.
• The transamination of oxaloacetate by glutamate to
yield aspartate and a-ketoglutarate is a prime example.
• There are two common process in biosynthesis of
amino acids
• Transamination – transfer of amine group
• One-carbon transfer
 Obtained from
glycolytic
• One-carbon transfer pathway
reaction also occurs
frequently in amino acid
biosynthesis Displacement of
electrons
• This can be seen in the
biosynthesis of amino
acids of the serine family
ie from serine to glycine
Transamination:
glutamate
donates nitrogen

Hydrolysis of
phosphate group

No C transfer
yet!!!!
 Observe C
transfer!!!

Produce amino acids in the serine family ; glycine and cysteine

– Glycine is generated by removal of the methyl group from serine

3 important carriers of one carbon units for this reaction

– Tetrahydrofolate- Carrier of methylene and formyl group
– Biotin - carrier of CO2
– S-adenosylmethionine- Carrier of methyl group
• Amino acids can also be placed in families based on biosynthetic
pathways
How does the biosynthesis of amino acids
related to carbohydrate metabolism?
Essential Amino Acids
• Biosynthesis of proteins require the presence of all the constituent
amino acids
• Missing amino acids will result in inhibition of protein biosynthesis
• Some organisms can synthesize all amino acids that they require e.g. E.
coli
• Other organism like human needs to get amino acids that we cannot
synthesize from food (diet) – essential amino acids
• Protein deficiency causes disease like kwashiorkor – not just starvation
but also breakdown of body’s own protein.
Feedback inhibition

• The biosynthetic pathway to produce amino acids and the bases of

nucleotides (purines and pyrimidines) are long, complex and high
energy requiring.
• So, cells have mechanism to stop biosynthesis if it is no longer
needed  feedback inhibition.
• Glycine, alanine and serine are key indicators of amino acid
metabolism in cells
• Access of any one single product can stop the biosynthetic pathway.
Amino acid catabolism
• What happens to access amino acids?
• Catabolism of amino acids
• Catabolism process
• Removal of nitrogen by transamination
• Transfer of N to α-ketoglutarate glutamate (leaving behind C-skeleton)
• C-skeleton:
• Glucogenic amino acid yeilds yields pyruvate /oxaloacetate / fumarate/ succinyl CoA / α
keto (starting point for production of glucose by gluconeogenesis)
• Glucogenic amino acid glutarate and later to glucose
• Ketogenic amino acid yields acetyl-CoA or acetoacetyl-CoA and later to Ketone Bodies

• Some amino acid are both ketogenic and glucogenic because

their carbon chain can form different products
Amino acid catabolism
• Fate of N: excreted in one of three forms
• Ammonia (ammonium ion) (e.g. fish)
• Urea (e.g. human)
• Uric acid (e.g. birds)
• Central pathway in nitrogen metabolism is the urea cycle.
The Urea Cycle
• Human central waste product of excess nitrogen is urea. Urea cycle
converts ammonia to urea
• Consist of 5 steps and took place in cytosol and mitochondria
• N and C in the guanidino group of Arg come from NH4+, HCO3-
(carbamoyl-P), and the a-NH2 of Glu and Asp
• Breakdown of Arg in the urea cycle releases two N and one C as
urea
• Liver catabolized most amino acids in terrestrial vertebrates
• Only ammonia in liver can be converted to urea: Urea is transported to
kidney where it will be eliminated
• Urea cycle is linked to TCA by fumarate  Krebs cycle
 The urea cycle series of reactions:
• A condensation reaction between the ammonium ion and
carbon dioxide produces carbomyl phosphate – require
hydrolysis of 2 ATP molecules for 1 molecule of carbomyl -P
• Transfer of the carbamoyl group of carbamoyl-P to ornithine
by ornithine transcarbamoylase (OTCase, reaction 1) yields
citrulline.
• The citrulline (transported to cytosol) ureido group is then
activated by reaction with ATP to give a citrullyl-AMP
intermediate (reaction 2a);
• AMP is then displaced by aspartate, which is linked to the
carbon framework of citrulline via its a-amino group (reaction
2b).
…urea cycle
• The product of this reaction is argininosuccinate; the enzyme catalyzing
the two steps of reaction 2 is argininosuccinate synthetase.
• The next step (reaction 3) is carried out by argininosuccinase, which
catalyzes the nonhydrolytic removal of fumarate from argininosuccinate
to give arginine. Hydrolysis of Arg by arginase (reaction 4) yields urea
and ornithine, completing the urea cycle.
*End of lecture*
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