Nitrogen fixation and assimilation

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Ch. 15 Nitrogen metabolism

• Nitrogen fixation and assimilation
• Available fixed nitrogen nitrate(NO3-), nitrite
  (NO2-), ammonia (NH4)
• Colonizing the root nodules of leguminous plants,
  some marine cyanobacteria
• Artificial nitrogen fixation 300-500oC, 300 atm
• Biological N2 reduction requires many ATP and a
  strong reducing agent, carried out by nitrogenase
  (inactivated by oxygen)

Figure 15.01 Root nodules from clover.
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• Other sources of fixed nitrogen
• nitrate(NO3-) naturally present nitrogen,
  reduced to ammonia (NH4) by plants and bacteria
• Nitrification
• Denitrification

Figure 15.03 The nitrogen cycle.
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• Assimilation of ammonia
• Glutamine synthetase
• ? a metabolic entry point for fixed nitrogen
• Glutamate ATP NH4 ? glutamine
  ADP Pi
• tightly regulated allosteric regulation (E.
  coli, dodecamer)
• Glutamate synthase provides glutamates for
  accepting amino group
• a -ketoglutarate glutamine NADPH ? 2
  Glutamate NADP
• Net result
• a-ketoglutarate NADPH ATP NH4
• ? Glutamate ADP Pi
  NADP

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• 2. Transamination
• Glutamate served as a amino-group donor
• Transaminase (fig 15.5)
• catalyze the transfer of an amino group to an
  a-keto acid
• ? Freely reversible, involved in synthesis and
  degradation of amino acids
• PLP(Pyridoxal-5-phosphate)
• ? a prosthetic group of pyridoxine to that the
  amino group is transiently attached
• ? Linked to eamino group of lysine
• Markers for damaged tissues SGOT, SGPT

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Figure 15.05 PLP-catalyzed transamination.
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• 3. Amino acid biosynthesis
• Synthesized from intermediates of glycolysis, the
  citric acid cycle, and the pentose phosphate
  pathway
• Amino group donor glutamate, glutamine
• 10 Essential amino acids

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Figure 15.06 Nitrogen metabolism in context.
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Synthesis of nonessential amino acids
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Tetrahydrofolate a carrier of one-carbon units,
vitamin
Figure 15.07 Tetrahydrofolate.
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• Synthesis of essential amino acids
• present only in microorgansims
•  considerably more complex than for nonessential
  amino acids
•   use familiar metabolic precursors
•   show species variation
• (1) Aspartate Family lysine,
  methionine,threonine
•     (2) Pyruvate Family leucine, isoleucine,
  valine
•     (3) Aromatic Family phenylalanine, tyrosine,
  tryptophan
•     (4) Histidine

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channeling movement of a reactant between two
active sites ex tryptophan synthase (a, b
subunit - indole)
Figure 15.08 Tryptophan synthase.
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(4) Histidine
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• 4. Amino acids as metabolic precursors
• Amino acids synthesized to proteins, converted
  to nucleotides, heme groups, neurotransmitters
• Many neurotransmitters are amino acid
  derivatives
• Neurotransmitters

Glutamate, Glycine GABA dopamine,
norepinephrine, epinephrine Serotonine
Prozac Melatonin NO (Box 15-A)
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• Nucleotides biosynthesis
• Synthesized from several amino acids
• No true dietary requirement efficient
  biosynthetic and salvage pathways

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Figure 15.09 AMP and GMP synthesis from IMP.
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Pyrimidine biosynthesis     UMP synthesis
feedback inhibition by UMP, UDP, UTP   
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• Synthesis of CTP from UTP

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Synthesis of dNTPs reduction of ADP, GDP, CDP,
UDP to dADP, dGDP, dCDP, dUDP by ribonucleotide
reductase (box 15.B)
Ribonucleotide reductase
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Synthesis of dTMP from dUMP
Thymidylate synthase dihydrofolate reductase vs
cancer          Anticancer agent inhibitor of
thymidylate synthase dihydrofolate reductase ,
eg 5-fluorodeoxyuridylate, methotrexate
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Thymidylate synthase dihydrofolate reductase
in one enzyme in plant and protozoa Figure 15.10
The bifunctional thymidylate synthase-dihydrofolat
e reductase of the protozoan Leishmania.
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• 5. Amino acids catabolism
• Amino acids as metabolic fuel
• Cells lining small intestine use dietary amino
  acids as E source
• liver aa originated from the diet, biosynthetic
  reactions and turnover of intracellular proteins
  (breakdown of muscle tissue)
• ? the catabolism of aa in the liver is not
  complete synthesized to glucose and exported to
  other tissues

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• Amino acids are glucogenic, ketogenic, or both
• glucogenic amino acids
• ketogenic amino acids
• Both glucogenic and ketogenic amino acids
• Table 15.2
• Ala, Asp, Glu converted to gluconeogenic
  substrates by transamination (Pyruvate, OAA,
  a-ketoglu)
• ? Asn, Gln, Ser deamination (Asp, glu, pyruvate)
• ? Pro, Arg, His converted to aketoglutarate via
  Glu
• ? Cys release ammonia and sulfur and converted
  to pyruvate

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• Amino acids are glucogenic, ketogenic, or both
• Table 15.2
• Ala, Asp, Glu converted to gluconeogenic
  substrates by transamination (Pyruvate, OAA,
  a-ketoglu)
• ? Asn, Gln, Ser deamination (Asp, glu, pyruvate)
• ? Pro, Arg, His converted to aketoglutarate via
  Glu
• Cys release ammonia and sulfur and converted to
  pyruvate
• ? Thr glucogenic and ketogenic and converted to
  glycine actyl-CoA
• ? Gly converted to methylene-tetrahydrofolate by
  glycine cleavage system (a multiprotein complex)
• ? Branched chain amino acids transamination,
  oxidative decarboxylation (fig 15.11)
• ? Aromatic amino acids glucogenic (alanine or
  fumarate) and ketogenic amino acids
  (acetoacetate)
• tetrahydrobiopterine

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Figure 15.11 The initial steps of valine
degradation.
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First step of phenylalanine degradation.
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Inborn errors of metabolism (Box 15.C)
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• 6. Nitrogen disposal The urea cycle
• Urea 80 of the bodys excess nitrogen is
  excreted as urea
• cf ammonia not a good form of disposal of
  excess nitrogen (neurotoxicity)
• Glutamate supplies nitrogen to the urea cycle
• Glutamate one of the most abundant amino acids
  inside cells
• Glutamate dehydrogenase mitochondrial enzyme,
  reversible
• ? Carbamoyl phosphate synthetase

The carbamoyl phosphate synthetase reaction.
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The urea cycle consists of four reactions Also
synthesized arginine (BUT essential?)
Figure 15.13 The four reactions of the urea
cycle.
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The urea cycle consists of four reactions
Figure 15.14 The urea cycle and related
reactions.
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• The urea cycle consists of four reactions
• Six enzymes mitochondrial and cytosolic,
• i) citrullinemitochondria ? ctyosol
• ii) ornithine ctyosol? mitochondria
• E consideration 4 ATP consumption/urea, but
  2NADH generation/urea ? 2 ATP gaining
• Regulation carbamoyl phosphate synthetase by
  N-acetylglutamate

Figure 15.14 The urea cycle and related
reactions.
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• Other mechanisms for nitrogen disposal
• Uric acids birds, reptile, WHY NOT UREA?
• Problem of uric acids kidney stone,
  precipitation in joint
• Excess pyrimidine bases can be degraded to CoA
  adducts
• ? Excess purine bases excreted
• Urease breaks down urea
• Urea H2O ? 2 NH3 CO2
• Present in bacteria, fungi
• ? The first enzyme to be crystalized (in 1926)

Figure 15.15 Urease.
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Uric acid is an end product of purine
catabolism - Catabolism and Salvage of Purine
Nucleotides