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Overview of Nitrogen Metabolismand Biosynthesis of Amino Acids
CH353 January 22, 2008
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The Nitrogen Cycle
• Nitrogen FixationN2 → NH4
+
• NitrificationNH3 → NO2
- → NO3-
• DenitrificationNO2
- & NO3- → N2
• Nitrogen Assimilation NH4
+ → Organic nitrogen
• Deamination
Organic nitrogen → NH4+
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Nitrogen Fixation
• Nitrogen Reduction Reaction
N2 + 3 H2 → 2NH3 ΔG′º = -33.5 kJ/mol – Exergonic reaction– High activation energy
– Stability of N2 triple bond D = 930 kJ/mol
• Biological Nitrogen ReductionN2 + 10H+ + 8e- + 16ATP → 2NH4
+ + 16ADP + 16Pi + H2
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Nitrogenase Complex
Dinitrogenase reductase• Dimer (Mr 60,000) of same subunits
• One 4Fe-4S between subunits• One ATP/ADP site per subunit• ATP binding enhances reduction
potential: E′º -300 → -420 mV
Dinitrogenase• Tetramer (Mr 240,000):
– 2 copies of 2 different subunits• 2 Mo, 32 Fe, 30 S per tetramer• 8 dinitrogenase reductase dimers each
transfers one e- to dinitrogenase
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Nitrogen Assimilation
• Glutamine SynthetaseGlutamate + NH4
+ + ATP → Glutamine + ADP + Pi + H+
• Glutamate Synthase (plants, bacteria)
α-Ketoglutarate + Glutamine + NADPH + H+ → 2 Glutamate + NADP+
• Net Reaction:α-Ketoglutarate + NH4
+ + NADPH + ATP → Glutamate + NADP+ + ADP + Pi
• Glutamate Dehydrogenase
α-Ketoglutarate + NH4+ + NADPH → Glutamate + NADP+
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Regulation of Glutamine Synthetase (E. coli)
Covalent Modification
Adenylylation inactivatesglutamine synthase
Adenylyltransferase(AT) regulated by PII
Uridylylation of PII
determines regulation:no UMP - adenylylationUMP – deadenylylation
α-Ketoglutarate & ATPstimulate uridylylation↑ Glutamine synthesis
Glutamine & Piinhibit uridylylation↓ Glutamine synthesis
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Regulation of Glutamine Synthetase (E. coli)
Transcription Activation• Uridylylated PII activates gene
encoding glutamine synthetase
Allosteric Inhibition• Glutamine synthetase is inhibited
by alanine, glycine and products of glutamine metabolism
• Binding of each metabolite causes partial inhibition; binding of all completely inhibits enzyme
• Overall effect of 8 inhibitors is more than additive: example of Concerted Inhibition
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Biosynthetic Reactions with Glutamine
Amidotransferase reactions• Glutamine is hydrolyzed to
glutamate + NH3 within the enzyme
• Acceptor hydroxyl or ketone often activated with ATP
Glutamine + Aspartate + ATP
↓Glutamate + Asparagine + AMP
+ PPi
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Overview of Amino Acid Biosynthesis
3-Phosphoglycerate Serine Glycine Cysteine
Pyruvate Alanine Valine Isoleucine Leucine
α-Ketoglutarate Glutamate Glutamine Proline Arginine
Ribose 5-phosphate Histidine
Erythrose-4-phosphatePhosphoenolpyruvate Tryptophan Phenylalanine Tyrosine
Oxaloacetate Aspartate Asparagine Methionine Lysine Threonine
nonessentialconditionalessential
Key:
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Reactions with Pyridoxal Phosphate
• Transamination (aminotransferase) reactions
Glutamate + Pyruvate α-Ketoglutarate + Alanine Glutamate + Oxaloacetate α-Ketoglutarate + Aspartate
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Amino Acids from α-Ketoglutarate
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Amino Acids from α-Ketoglutarate
α-Ketoglutarate
Glutamate Glutamine
Glutamate -semialdehyde
Arginine
Proline
Ornithine
Urea Cycle
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Biosynthesis of Proline and Arginine
ornithineaminotransferase
Glutamate α-Ketoglutarate
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Amino Acids from 3-Phosphoglycerate
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Biosynthesis of Serine and Glycine
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Cofactors for One-Carbon Metabolism
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One-Carbon Units on Tetrahydrofolate
• tetrahydrofolate (H4 folate) is derived from folic acid
• one-carbons on H4 folate can have 3 redox states
• hydroxymethyl group of serine and formate are primary entry metabolites
• reversible conversions except for production of N 5-methyl H4 folate
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The Activated Methyl Cycle
• S-Adenosyl methionine is the methyl donor for nearly all reactions
• Methyl group of methionine is replenished with N5-methyl H4 folate
• Methionine synthase requires coenzyme B12 (from vitamin B12)
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Biosynthesis of Cysteine
• Only bacteria and plants can assimilate inorganic sulfur
• In mammals, cysteine is conditionally essential deriving its sulfur from methionine
• Cysteine is biosynthesized from serine and homocysteine, a methionine metabolite
• Sulfhydryl is transferred from homocysteine to serine in two PLP dependent steps with cystathionine as intermediate
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Study Problem
• High serum homocysteine level is a risk factor for coronary heart disease and arteriosclerosis
• Some individuals with high homocysteine have variations in the cystathionine β-synthase gene
• A combination of vitamin supplements is recommended for alleviating high homocysteine
• Based upon the pathways for homocysteine synthesis and utilization, which 3 vitamins would you recommend?
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Amino Acids from Oxaloacetate and Pyruvate
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Amino Acids from Oxaloacetate
Oxaloacetate
Aspartate Asparagine
Pyruvate
Lysine
Aspartate β-semialdehyde
Threonine Methionine
HomoserineDihydropicolinate
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Biosynthesis of Lysine, Methione & Threonine
Lysine Methionine & Threonine
Dihydropicolinate
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Biosynthesis of Methionine
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Biosynthesis of Methionine
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Homocysteine Serine Cysteine Homoserine
Cystathionine
H2Ocystathionine β-synthase
H2Ocystathionine
γ-synthase
H2O cystathionine β-lyase
H2Ocystathionine γ-lyase
α-Ketobutyrate Cysteine Pyruvate Homocysteine
Biosynthesis of Cysteine Biosynthesis of Methionine
γ βPLP PLP
PLP PLP
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Biosynthesis of Threonine
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Amino Acids from Pyruvate
Alanine
Valine Leucine Isoleucine
Pyruvate
Pyruvate
CO2
α-Ketobutyrate
Threonine
acetolactatesynthetase
Acetyl-CoA
CO2
Same enzymes for both valine and isoleucine
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Biosynthesis of Isoleucine and Valine
Isoleucine Valine & Leucine
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Regulation of Amino Acid Biosynthesis
Multiple Isozymes for• Aspartokinase (A1, A2, A3)
• Homoserine dehydrogenase (B1, B2)
• Threonine dehydratase (C1, C2)
Allosteric regulation of selective isozymes – some unregulated
Sequential feedback inhibition• Same product inhibits its biosynthetic
path at multiple sites
Inhibits first enzyme in pathway
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Biosynthesis of Aromatic Amino Acids
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Biosynthesis of Aromatic Amino Acids
Phosphoenolpyruvate Erythrose 4-phosphate
Shikimate
Chorismate
Prephenate
CO2CO2
Tyrosine Phenylalanine
Anthranilate
Pyruvate
PRPP
CO2 Serine
Glyceraldehyde 3-phosphate
Tryptophan
Glu Gln
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Conversion of Phenylalanine to Tyrosine
• Tyrosine is a conditionally essential amino acid
• It can be synthesized from phenylalanine as part of its catabolic pathway
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Biosynthesis of Histidine
PRPP ATP
Histidine
AICARPurineBiosynthesis
Glutamine
Glutamate