1. Overview of Nitrogen Metabolism and Biosynthesis of Amino Acids
CH353 January 22, 2008

2. The Nitrogen Cycle Nitrogen Fixation Nitrification Denitrification
N2 → NH4+
Nitrification
NH3 → NO2- → NO3-
Denitrification
NO2- & NO3- → N2
Nitrogen Assimilation
NH4+ → Organic nitrogen
Deamination
Organic nitrogen → NH4+

3. N2 + 10H+ + 8e- + 16ATP → 2NH4+ + 16ADP + 16Pi + H2
Nitrogen Fixation
Nitrogen Reduction Reaction
N2 + 3 H2 → 2NH3 ΔG′º = kJ/mol
Exergonic reaction
High activation energy
Stability of N2 triple bond D = 930 kJ/mol
Biological Nitrogen Reduction
N2 + 10H+ + 8e- + 16ATP → 2NH ADP + 16Pi + H2

4. Nitrogenase Complex Dinitrogenase reductase Dinitrogenase
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 → -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

5. Nitrogen Assimilation
Glutamine Synthetase
Glutamate + 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+

6. Regulation of Glutamine Synthetase (E. coli)
Covalent Modification
Adenylylation inactivates
glutamine synthase
Adenylyltransferase
(AT) regulated by PII
Uridylylation of PII
determines regulation:
no UMP - adenylylation
UMP – deadenylylation
α-Ketoglutarate & ATP
stimulate uridylylation
↑ Glutamine synthesis
Glutamine & Pi
inhibit uridylylation
↓ Glutamine synthesis

7. 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

8. 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

9. Overview of Amino Acid Biosynthesis
Ribose 5-phosphate
Histidine
3-Phosphoglycerate
Serine
Glycine
Cysteine
Erythrose-4-phosphate
Phosphoenolpyruvate
Tryptophan
Phenylalanine
Tyrosine
Pyruvate
Alanine
Valine
Isoleucine
Leucine
Oxaloacetate
Aspartate
Asparagine
Methionine
Lysine
Threonine
α-Ketoglutarate
Glutamate
Glutamine
Proline
Arginine
Key:
nonessential
conditional
essential

10. Reactions with Pyridoxal Phosphate
Transamination (aminotransferase) reactions
Glutamate + Pyruvate α-Ketoglutarate + Alanine
Glutamate + Oxaloacetate α-Ketoglutarate + Aspartate

11. Amino Acids from α-Ketoglutarate

12. Amino Acids from α-Ketoglutarate
Glutamate
Glutamine
Glutamate g-semialdehyde
Arginine
Proline
Ornithine
Urea Cycle

13. Biosynthesis of Proline and Arginine
ornithine
aminotransferase
Glutamate α-Ketoglutarate

14. Amino Acids from 3-Phosphoglycerate

15. Biosynthesis of Serine and Glycine

16. Cofactors for One-Carbon Metabolism

17. 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

18. 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)

19. 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

20. 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?

21. Amino Acids from Oxaloacetate and Pyruvate

22. Amino Acids from Oxaloacetate
Aspartate
Asparagine
Pyruvate
Aspartate β-semialdehyde
Dihydropicolinate
Homoserine
Lysine
Methionine
Threonine

23. Biosynthesis of Lysine, Methione & Threonine
Dihydropicolinate
Lysine
Methionine & Threonine

24. Biosynthesis of Methionine

25. Biosynthesis of Methionine

26. Biosynthesis of Cysteine Biosynthesis of Methionine
Homocysteine
Serine
Cysteine
Homoserine
H2O
cystathionine β-synthase
cystathionine γ-synthase
γ β
PLP
Cystathionine
H2O
cystathionine β-lyase
cystathionine γ-lyase
PLP
α-Ketobutyrate
Cysteine
Pyruvate
Homocysteine

27. Biosynthesis of Threonine

28. Amino Acids from Pyruvate
Threonine
Pyruvate
Alanine
CO2
α-Ketobutyrate
acetolactate
synthetase
Pyruvate
Acetyl-CoA
Same enzymes for both valine and isoleucine
CO2
Isoleucine
Valine
Leucine

29. Biosynthesis of Isoleucine and Valine
Valine & Leucine

30. 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

31. Biosynthesis of Aromatic Amino Acids

32. Biosynthesis of Aromatic Amino Acids
Phosphoenolpyruvate
Erythrose 4-phosphate
Shikimate
Pyruvate
Glu Gln
Chorismate
Anthranilate
PRPP
Prephenate
CO2
CO2
Serine
CO2
Tyrosine
Phenylalanine
Glyceraldehyde 3-phosphate
Tryptophan

33. Conversion of Phenylalanine to Tyrosine
Tyrosine is a conditionally essential amino acid
It can be synthesized from phenylalanine as part of its catabolic pathway

35. Biosynthesis of Histidine
PRPP
ATP
Glutamine
Purine
Biosynthesis
AICAR
Glutamate
Histidine