Amino Acid Metabolism:

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• Amino Acid Metabolism
• Nitrogen Assimilation
• and Amino Acid Biosynthesis

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Nitrogen Assimilation

• Nitrogen is required in the synthesis of amino
  acids, purine and pyrimidine nucleotides, and a
  number of other important biological compounds.
• Organisms need to obtain nitrogen in a usable
  form. Nitrogen in the form of ammonia is
  assimilated by biological systems
• Nitrogen is originally assimilated from the
  environment by microorganisms and plants.
• Animals must obtain biological forms of nitrogen
  from their diets.

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Nitrogen Fixation

• Most abundant form of nitrogen on Earth is N2 gas
  (makes up 80 of air)
• N2 gas is very stable and inert. 2 N connected by
  triple bond (225 kcal/mole required to break
  bond).
• N2 gas can be converted to biologically
  accessible forms in three ways
• N2 can be reduced to NO3- by lightning and UV
  radiation (15 of fixed nitrogen)
• N2 can be reduced to NH3 through industrial
  processes (25 of total fixed nitrogen) Requires
  temperatures of 500oC and 300 atm)
• N2 can be reduced to NH3 by nitrogen fixing
  bacteria (60 0f total fixed nitrogen)

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Nitrogen Cycle
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Biological Nitrogen Fixation

• Process performed only by special free living
  (cyanobacteria) microorganims.
• Nitrogen fixation can also be performed by
  microorganisms (Rhizobium, Bradyrhizobium) that
  exist as symbiotes with specific plant species
  (Legumes soybean, alfalfa)
• N2 is converted to NH3 in a reaction catalyzed by
  the nitrogenase complex.

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Nitrogenase

• consist of two subunits, an Fe-protein
  (reductase) and an MoFe-protein (nitrogenase).
• Reductive process N2 8e- 8H ? 2 NH3 H2
• For every e- used in the process 2 ATP are
  consumed, so 16 ATPs are required to convert 1 N2
  to NH3
• Enzymes are highly sensitive to O2. Require
  anerobic conditions

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Nitrate and Nitrite to Ammonia

• NO3- and NO2- must be converted to NH3 to be
  assimilated into organisms.
• Process referred to as nitrification
• Requires two enzymes nitrate reductase and
  nitrite reductase

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Ammonia Assimilation

• Glutamate dehydrogenase - reductive amination of
  a-ketogluturate to glutamate.
• Glutamine synthetase - ATPdependent amidation of
  gamma-carboxyl of glutamate to glutamine

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Glutamate Dehydrogenase

• Impt. in bacteria when ammonia is present at high
  concentrations.
• In animal and plant systems enzyme is localized
  within the mitochondria. The enzyme is near
  equilibrium and runs in the direction toward
  glutamate catabolism (anaplerotic rxn to
  regenerate a-ketogluturate).

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Glutamine Synthetase (GS)

• Glutamine plays important role in N-metabolism.
• Acts a NH3 donor for purine and pyrimidine
  synthesis
• In mammals, glutamine is synthesized in muscle
  tissue using GS and transported to other tissues.

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Glutamine back to Glutamate

• Under low NH3 conditions, in conjunction with
  glutamine synthetase, bacteria use glutamate
  synthase to convert Gln and a-ketogluturate to 2
  glutamates.
• The Km of glutamate dehydrogenase for NH3 is
  high, the Km of glutamine synthetase is low.

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Glutamine Synthetase

• GS in bacteria is regulated in three ways
• Feedback inhibition
• Covalent modification (interconverts between
  inactive and active forms)
• Regulation of gene expression and protein
  synthesis control the amount of GS in cells

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Allosteric Modulation of GS Acitivity in Bacteria
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Allosteric Modulators of Mammalian GS

• Activator a-ketogluturate
• Inhibitors glycine, serine, alanine,
  carbamyl-phosphate

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Regulation of Bacterial GS by Covalent
Modification
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• Gene GlnA is actively transcribed only if
  transcriptional enhancer NRI is in its
  phosphorylated form, NRI-P
• NRI is phosphorylated by NRII, a protein kinase
• If NRII is complexed with the activated form of
  PII acts as a phosphatase, not a kinase

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• Glutamate serves as primary N- donor for AA
  synthesis through transamination reactions

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Transamination rxns involve Pyridoxol-phosphate
and formation of Schiff Base
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Essential vs. Non-Essential AAs

• Plants and bacteria can synthesize all 20 amino
  acids.
• Animals must obtain 8 amino acids (essential
  AAs) from diet

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Amino Acid Biosynthesis is a Diverse Process
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