1. Andy Howard Biochemistry Lectures, Spring 2019 Thursday 25 April 2019
Nitrogen Metabolism 1
Andy Howard Biochemistry Lectures, Spring 2019 Thursday 25 April 2019

2. Nitrogen metabolism
Amino acid anabolism sometimes involves little more than transamination, but often is more complex
Amino acid catabolism feeds the TCA cycle and acetyl CoA
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3. What we’ll discuss Nitrogenase Nitrogen cycles AA anabolism
Glutamate
Transaminations
Simple syntheses
Complex syntheses
AA catabolism
Transaminations
Glucogenic & Ketogenic aa’s
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4. The nitrogen pool
Nitrogen fixation from air (N2  NH3) doesn’t produce a large percentage of circulating biological nitrogen but it’s the ultimate source of most of it
Other entries in pool: nitrate (NO3 -), nitrite (NO2-)
Most of this difficult biochemistry is bacterial
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5. Nitrogenase
Enzyme found in Rhizobium, a bacterium that colonizes & lives symbiotically in the root nodules of legumes and a few other plants
Also in free-living microorganisms like Azotobacter
Energetically expensive but irreversible path to reduction of dinitrogen to ammonia:
N2 + 8H+ + 8e ATP  2NH3 + H2 + 16ADP + 16Pi
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6. Nitrogenase structure
Multi-component complex
Mo-Fe active site in actual N2-fixing component
Azotobacter Nitrogenase Mo-Fe, Fe proteins 350 kDa heterooctamer EC PDB 1G20, 2.2Å
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7. Mechanistic intermediates
Probably proceeds via diimine and hydrazine:
NN + 2e- + 2H+  H-N=N-H
H-N=N-H + 2e- + 2H+  H2N-NH2
H2N-NH2 + 2e- + 2H+  2 NH3
2e- + 2H+  H2 
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8. Ammonia, nitrate, nitrite
Ammonia comes from decayed organisms and is oxidized in soil bacteria to nitrate (nitrification)
Nitrate reductase and nitrite reductase found in plants and microorganisms
Alcaligines
Nitrite
Reductase 111 kDa trimer monomer shown PDB 2BO0, 1.35Å
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9. Reductase reactions NO3- + 2e- + 2H+  NO2- + H2O
NO2- + 6e- + 7H+  NH3 + 2 H2O
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10. Essential and non-essential amino acids
An amino acid is defined as essential if it must be obtained within the diet
In general the essential amino acids are the ones that have complicated and highly ATP-dependent biosynthetic pathways
Of course, it depends on the organism
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11. Essential & non-essential aa’s
A.A. name # ATP’s
Glycine 12
Serine 18
Cysteine 19
Alanine 20
Aspartate 21
Asparagine 22-24
Glutamate 30
Glutamine 31
Proline 39
Arginine 44 *
Tyrosine 62 **
Essential
A.A. Name #ATPs
Threonine 31
Valine 39
Histidine 42
Methionine 44
Leucine 47
Lysine 50-51
Isoleucine 55
Phenylalanine 65
Tryptophan 78
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12. Transaminations
General process of interconverting -amino acids and -ketoacids
Primary way that N gets incorporated into non-N-containing structures
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13. Reaction dynamics
All (?) transaminations involve PLP as a cofactor: see mechanism in textbook
These are actually oxidation-reduction reactions, since we’re swapping an amine (carbon oxidation state +2) for a carbonyl (carbon oxidation state 0)
But there is no external oxidizing agent
E.coli Aspartate aminotransferase
EC kDa dimer;
Monomer shown PDB 2Q7W, 1.4Å
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14. Examples of transaminases
Reactants Products Trans- Keto acid amino acid keto acid amino acid aminase
Pyruvate glutamate -k-glutarate alanine pyruvate
Pyruvate aspartate oxaloacetate alanine pyruvate
Oxaloacetate glutamate -k-glutarate aspartate aspartate
3-phosphono- glutamate -k-glutarate phosphoserine phospo- hydroxypyruvate serine
4-OH-phenyl- glutamate -k-glutarate tyrosine tyrosine pyruvate
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15. Catabolic or anabolic?
From the point of view of available pools of amino acids, these are amphibolic:
They involve synthesis of one amino acid at the expense of another
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16. iClicker question #1
1. The distinction between essential and non-essential amino acids relates to
(a) whether an organism can synthesize the amino acid
(b) how we degrade the amino acid
(c) how many kJ/mol can be derived from its catabolism
(d) how many sulfurs are present
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17. Biosynthetic pathways to specific amino acids
Some are complex and energy-requiring
Can be logically divided according to chemical properties of the target amino acids:
Small
Branched-chain aliphatic
Neutral polar
Acidic
Basic
Aromatic
Sulfur-containing
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18. Which amino acids in which categories?
Category Amino acids
Small gly, ala, ?pro?
Branched-chain val, leu, ile
Neutral polar asn, gln, ser, thr
Acidic asp, glu
Basic lys, arg
Aromatic phe, tyr, trp, his
Sulfur-containing cys, met
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19. Glutamate
Glutamate is a critical metabolite because so many of the transaminations start with it as the amine donor
It is produced in E.coli, etc. via glutamate dehydrogenase using ammonium ion as nitrogen donor:
Glu dehydrogenase
PDB 1BGV 296 kDa hexamer monomer shown
EC , 1.9Å Clostridium
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20. Glutamate dehydrogenase reaction
-ketoglutarate + NH4+ + NAD(P)H + H+  NAD(P)+ + H2O + glutamate
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21. Glutamine
Glutamate can be aminated with expenditure of ATP to form glutamine: glutamate + NH4+ + ATP  glutamine + ADP + Pi
Note that glutamine synthetase is a ligase: the ATP is an energy-provider, not a phosphate donor
Human Glutamine synthetase
EC kDa pentamer PDB 2OJW, 2.05Å
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22. Aspartate and asparagine
Asp is simple: transamination of oxaloacetate
Asn is straightforward too
asparagine synthetase moves amine from gln to asp, leaving glu (another ligase)
Gln + asp + ATP  AMP + PPi + glu + asn
E.coli Asparagine synthetase B
EC kDa tetramer
PDB 1CT9, 2Å
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23. Simple: ala, gly, ser Alanine by transamination from pyruvate
Glycine from serine by SHMT (q.v.)
Serine from 3-phosphoglycerate:
3-phosphoglycerate + NAD+  NADH + H+ + 3-phosphohydroxypyruvate
3-phosphohydroxypyruvate + glutamate  3-phosphoserine + -ketoglutarate
3-phosphoserine + H2O  serine + Pi
Human Phosphoserine phosphatase 49 kDa dimer EC PDB 1NNL,1.53Å
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24. Serine hydroxymethyl- transferase
Serine + tetrahydrofolate  H2O + glycine + 5,10-methylene- tetrahydrofolate
This can be viewed as a source of methylene units for other biosyntheses
PLP-dependent reaction
Thermus thermophilus
SHMT 90 kDa dimer EC
PDB 2DKJ,1.15Å
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25. Arginine & proline Two routes: Glutamate to glutamate semialdehyde
that cyclizes to 1-pyrroline 5-carboxylate and thence to proline
Glutamate semialdehyde can also be converted to ornithine and thence to arg
Alternative: glutamate acetylated to N-acetyl-glutamate-5-semialdehyde and thence to ornithine
ornithine
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26. Glutamate to P5C
Single enzyme can interconvert glutamate and 1-pyrroline carboxylate: 1-pyrroline-5-carboxylate dehydrogenase
3-layer  sandwich protein
Thermus thermophilus PCD 300 kDa hexamer dimer shown EC
PDB 2BJA, 1.9Å
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27. Pyrroline-5-carboxylate to proline
Pyrroline-5-carboxylate reduced to proline
Large, NAD(P)H-dependent enzyme
Human Pyrroline-5-carboxylate reductase EC kDa decamer pentamer shown PDB 2IZZ, 1.95Å
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28. Glutamate to Glu semialdehyde
Glu is -phosphorylated: glu + ATP  glu-5-P +ADP ( )
Glu-5-P is reduced and dephosphorylated: glu-5-P + NADPH + H+  glu-5-semialdehyde + NADP+ + Pi
Thermatoga maritima
-glutamyl phosphate reductase 47 kDa monomer EC PDB 1O20, 2Å
Glu-5-P
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29. Glu semialdehyde to ornithine
This is just another transamination, catalyzed by ornithine aminotransferase: glu-5-semialdehyde + glu/asp  ornithine + -ketoglutarate / oxaloacetate
Typical PLP-dependent reaction
Human OAT 193 kDa tetramer EC PDB 2OAT, 1.95Å
ornithine
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30. Ornithine to citrulline
Carbamoyl phosphate
Ornithine to citrulline
Ornithine condenses with carbamoyl phosphate to form citrulline with the help of ornithine transcarbamoylase
110 kDa trimer E.coli EC , PDB 1DUV, 1.7Å
citrulline
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31. Citrulline to argininosuccinate
Citrulline condenses with aspartate using ATP hydrolysis to drive it forward to L-argininosuccinate: citrulline + aspartate + ATP  L-argininosuccinate + AMP + PPi
Argininosuccinate synthase PDB 2NZ2, 2.4Å EC
200 kDa tetramer monomer shown
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32. Argininosuccinate to arginine
Fumarate extracted, leaving arginine
Argininosuccinate lyase is also -crystallin, one of the moonlighting proteins: it’s a component of eye lenses
E.coli ASL 100 kDa dimer EC , 2.44Å PDB 1TJ7, 2.44
fumarate
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33. Why all that detail?
These reactions form 75% of the urea cycle, which is an important path for amino acid and nucleic acid degradation.
So we’ll need this later.
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34. Cysteine synthesis in plants and bacteria
serine + Acetyl CoA  O-acetylserine + HSCoA
O-acetylserine + S2- + H+  cysteine + acetate
Ser acetyltransferase is inhibited by cysteine
Haemophilus serine acetyltransferase EC kDa hexamer dimer shown PDB 1SSQ, 1.85Å
O-acetylserine
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35. Animal pathway to cys
Ser + homocysteine (from met) fuse to form cystathionine + H2O
Cystathionine + H2O  NH4+ + cysteine + -ketobutyrate
Yeast
Cystathionine -lyase EC PDB 1N8P, 2.6 Å
173 kDa tetramer
cystathionine
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36. Marching through the list of twenty amino acids
Amino acids we’ve already covered
Acids and amides: glu, gln, asp, asn
Simple: ala, ser, gly
Others: arg, pro, cys
Essential but straightforward
met, thr
val, leu, ile
Essential & Ugly: lys, phe, tyr, trp, his
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37. Lys, met, thr
asp gets phosphorylated and becomes a source for all of these:
asp + ATP -aspartyl phosphate + ADP via aspartate kinase
-asp P + NADPH + H+  Pi + aspartate -semialdehyde +NADP+
This heads to lys or to homoserine
Homoserine converts in a few steps to met or thr
Arabidopsis Aspartate kinase 112 kDa dimer EC PDB 2CDQ, 2.85Å
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38. Asp -semialdehyde to homoserine
-aldehyde reduced to sec-alcohol, which is homoserine
Homo is generally a prefix meaning containing an extra methylene group
This is precursor to homocysteine  methionine
It also leads to threonine
homoserine
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39. Homoserine to threonine
Phospho- homoserine
Homoserine to threonine
Homoserine phosphorylated with ATP as phosphate donor
Phosphohomoserine dephosphorylated with movement of -OH from one carbon to another: threonine results
threonine
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40. Homoserine to met Three reactions convert homoserine to homocysteine
5-methyltetrahydrofolate serves as a methyl donor to convert homocysteine to methionine via methionine synthase
This enzyme exists in humans but its activity is low and [homocysteine] is low;
So methionine is essential in humans
methionine
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41. 2,3-dihydro- picolinate
Lysine I
2,3,4,5-tetrahydro-picolinate
Aspartyl semialdehyde condenses with pyruvate to form 2,3-dihydropicolinate
Reduced again to 2,3,4,5- tetrahydropicolinate
Acylated (via AcylCoA) to N- acyl-2-amino-6-oxopimelate
N-succinyl- 2-amino-6-oxopimelate
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42. Lysine II
N-acyl-2-amino-6-oxopimelate transaminated to N-acyl-2,6-diaminopimelate
Deacylated to L,L-2,6-diaminopimelate
Epimerase converts that to meso form
That’s decarboxylated to lysine
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43. What’s left? (= “done”)
Non-essential
A.A. name # ATP’s
Glycine 12
Serine 18
Cysteine 19
Alanine 20
Aspartate 21
Asparagine 22-24
Glutamate 30
Glutamine 31
Proline 39
Arginine 44 *
Tyrosine 62 **
Essential
A.A. Name #ATPs
Threonine 31
Valine 39
Histidine 42
Methionine 44
Leucine 47
Lysine 50-51
Isoleucine 55
Phenylalanine 65
Tryptophan 78
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44. Branched-chain aliphatics: isoleucine and valine
-ketobutyrate
Derived from pyruvate or - ketobutyrate
2 pyruvate  -ketoisovalerate + CO2
pyr + -ketobutyrate  -keto--methylvalerate + CO2
These products are transaminated to val and ile
α-keto-isovalerate
-keto -methylvalerate
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45. Leucine Also derived from -ketoisovalerate;
An extra methylene is inserted between the polar end and the isopropyl group
Final reaction is another transamination
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46. Aromatics: phe and tyr shikimate
Common pathways for phe,tyr,trp via shikimate and chorismate
For phe, tyr: chorismate converted to prephenate
Prephenate can be aromatized with or without a 4-OH group to lead to phe,tyr
chorismate
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47. prephenate
Reaction specifics
Prephenate is oxidized and dehydroxylated in two steps to phenylpyruvate
Or it is oxidized to 4-OH- phenylpyruvate
Transaminations of those - ketoacids yield the final amino acids
4-hydroxy- phenyl- pyruvate
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48. Chorismate mutase Isomerase, converts chorismate to prephenate
In E.coli: 2 versions depending on which path the product is heading to
Active sites are similar in all organisms but architecture is very different
Catalytic triad similar to serine proteases
B.subtilis chorismate mutase 42 kDa trimer EC
PDB 1DBF, 1.3Å
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49. Histidine PRPP Start with PRPP and ATP: form phosphoribosyl ATP
3 reactions involving glutamine as nitrogen donor for ring lead to imidazole glycerol phosphate
That gets modified and transaminated to make histidine
imidazole glycerol phosphate
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50. Tryptophan Also via chorismate
anthranilate
Also via chorismate
Sidechain amide transferred to chorismate
Conversion to anthranilate
PRPP provides phosphoribosyl moiety; this sets up indole glycerol phosphate
Tryptophan synthase eliminates glyc-P, adds ser
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51. iClicker question #2
2. Which of these amino acids is not synthesized through a pathway involving chorismate?
(a) phe
(b) his
(c) trp
(d) tyr
(e) all of those involve chorismate
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52. What do we do with amino acids?
Obviously a lot of them serve as building-blocks for protein and peptide synthesis via ribosomal mechanisms
Also serve as metabolites, getting converted to other compounds (including nucleic acids) or getting oxidized as fuel
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53. Transaminations Generally two stages:
amino acid + -ketoglutarate  -keto acid + glutamate
Glutamate + NAD+ + H2O  -ketoglutarate + NADH + H+ + NH4+
Net reaction is amino acid + NAD+ + H2O  -keto acid- + NADH + H+ + NH4+
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54. Glucogenic amino acids
Degradation of many amino acids lead to TCA cycle intermediates or pyruvate
These can be built back up to glucose;
So these are called glucogenic
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55. Ketogenic amino acids
Degradation of others leads to acetyl CoA and related compounds
these cannot be built back up to glucose except via the glyoxalate shuttle
these are called ketogenic
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56. Glucogenic amino acids
Amino acids that can be catabolized to produce building blocks that lead to glucose without help of glyoxalate pathway
Most produce pyruvate, -ketoglutarate, succinyl CoA, succinate, fumarate, or oxaloacetate
By convention, amino acids that produce both TCA-cycle intermediates and acetyl CoA are labeled as glucogenic
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