Chapter 18- Amino Acid and Nitrogen Metabolism

All compounds can convert…

• Ammonia (NH3) to organic nitrogen (compounds containing C-N bonds)

• This is because, there is much more nitrogen available as dinitrogen gas (n2); however, far fewer organisms can synthesize NH3 from N2 (biological nitrogen fixation)

• Conversion of NO3- (nitrate) to NH3 is widespread among plants and microorganisms, but many soils are NO3 poor

Nitrogen availability limits…

• growth for most organisms

Relationship between inorganic and organic nitrogen metabolism Pathway 1

N2 (inorganic)→ NH3 (Inorganic)→AA/nucleotides,coenzymes,porphryins→ Proteins/DNA/RNA/COMPLEX POLYSACC/PHOSPHOLIPIDS

Relationship between inorganic and organic nitrogen metabolism Pathway 2

N2 (inorganic)→NH3 (inorganic)→energy→N02- Nitrate(inorganic)→energy→NO3- nitrate (inorganic)

Nitrogen fixation —

converts N₂ → NH₃ (only bacteria & archaea do this)

Nitrification —

NH₃ → NO₂⁻ → NO₃⁻

Assimilation —

• plants & microbes convert NH₃/NO₃⁻ → organic

Denitrification

— NO₃⁻ → N₂ (returns N₂ to atmosphere)

Inorganic N:

N₂, NH₃, NO₂⁻, NO₃⁻

Organic N:

amino acids, nucleotides, etc

Nitrogen Fixation — Nitrogenase Reaction

• reducing atmospheric N₂ → NH₃ (ammonia).

• Occurs in symbiotic bacteria (e.g., Rhizobium in soybean root nodules).

• Industrial Haber-Bosch process

The nitrogenase reaction to memorize:

N2​+ 8H+ +8e− +16MgATP→2NH3​+H2​+16MgADP+16Pi

Important notes nitrogenase reaction

• Huge ATP cost (16 ATP!)

• Requires strong reducing power

• Carried out by molybdenum-dependent nitrogenase

• Electrons flow from reduced ferredoxin/flavodoxin to nitrogenase complex

Importance of Nitrogenase Reaction

Nitrogenase makes ammonia, which becomes the starting point for all organic nitrogen compounds in living systems.

Once you’ve made ammonia (NH₃), you need to… 

• trap it safely, because free ammonia is toxic.

• Cells do this by converting NH₃ into four major organic carriers

Ammonia Assimilation — The 4 Key Organic N Products (Fig 18.5)

• Glutamate — via glutamate dehydrogenase (GDH)

• Glutamine — via glutamine synthetase (GS)

• Asparagine — via asparagine synthase

• Carbamoyl phosphate — via carbamoyl phosphate synthetase (CPS)

Glutamate —

• via glutamate dehydrogenase (GDH)

• Reaction:
  α-ketoglutarate + NH₃ + NAD(P)H ⇌ glutamate

  Key points:

  • This is a reductive amination (adding NH₃ to a carbonyl).

  • Extremely important in bacteria (main glutamate-producing pathway).

  • In animals, the reaction tends to go reverse (glutamate → α-KG + NH₃) because NH₃ is low inside cells.

Glutamine —

• via glutamine synthetase (GS)

• Reaction:
  Glutamate + NH₃ + ATP → Glutamine + ADP + Pi

  Key points:

  • Uses ATP (that’s why it's a synthetase).

  • The amide nitrogen of glutamine becomes a “donor nitrogen” used everywhere — nucleotides, amino acids, etc.

  • One of the most regulated enzymes in all biology.

Asparagine —

• via asparagine synthase

• Glutamine donates its amide N to convert:
  Aspartate → Asparagine

  Key points:

  • Mechanistically similar to GS.

  • Uses ATP to activate Asp first.

  • Asparagine is used in protein synthesis and nitrogen storage/transport

Carbamoyl phosphate —

• via carbamoyl phosphate synthetase (CPS)

• Reaction:
  NH₃ (or glutamine) + CO₂ + 2 ATP → carbamoyl phosphate

Key points:

• Carbamoyl phosphate is used for:

  • Urea cycle (to detoxify ammonia)

  • Pyrimidine synthesis

• Needs 2 ATP

• Activated by N-acetylglutamate in the urea cycle

This enzyme is a huge deal in nitrogen detoxification.

Why degrade proteins?

Cells constantly replace proteins to:

• Remove damaged or misfolded proteins

• Regulate levels of enzymes

• Recycle amino acids
  Most proteins have a half-life of 1–2 days.

The Ubiquitin–Proteasome System (UPS) Step 1

76-aa protein attached to lysine residues on target proteins.

Attachment occurs via an isopeptide bond between:

• Ubiquitin’s C-terminal Gly, and

• Lysine amino group on the target protein

✨ This process requires ATP to activate ubiquitin.

Ubiquitin–Proteasome System (UPS) Step 1 Enzymes

Enzymes required:

1. E1 – Ubiquitin‐activating enzyme

2. E2 – Ubiquitin‐conjugating enzyme

3. E3 – Ubiquitin ligase ← the specific targeting enzyme

   • Recognizes the protein to be degraded

   • Attaches ubiquitin

   • Builds polyubiquitin chains

Polyubiquitin = the "DEGRADE ME" signal.

Ubiquitin–Proteasome System (UPS) Step 2

Recognition by the proteasome

The 20S proteasome is a large ATP-dependent protease complex.
Polyubiquitinated proteins are fed into it → broken down into small peptides.
ATP is needed for:

• Opening the proteasome cap

• Unfolding the target protein

The proteasome is cytosolic and extremely tightly regulated.

What PLP, Pyridoxal Phosphate, does: 5

PLP assists reactions at the α-, β-, or γ-carbon of amino acids, including:

• Transamination (most important for nitrogen metabolism)

• Decarboxylation

• Racemization

• Eliminations

• Retro-aldol reactions

Mechanism you must know Pyridoxal Phosphate

PLP forms a Schiff base with the amino acid substrate → stabilizes carbanion intermediates.v

Tetrahydrofolate

• THE one-carbon transfer coenzyme.

• Single carbon units in different oxidation states:

  • Methyl (–CH₃)

  • Methylene (–CH₂–)

  • Formyl (–CHO)

  These carbon units are used for:

  • Nucleotide synthesis

  • Methionine regeneration

  • C–C and C–N bond formation

• Key vitamin:

  Derived from folate (Vitamin B9).
  Deficiency → neural tube defects + cardiovascular disease.

Vitamin B₁₂ — Cobalamin

Used only in a small number of reactions, but essential.

Two active forms:

• Methyl-B12 → methionine synthase

• Adenosyl-B12 → methylmalonyl-CoA mutase

Medical point you must know:

Pernicious anemia
= autoimmune destruction of intrinsic factor → can't absorb B12 → deficiency.

1. First Step of Amino Acid Degradation: Transamination

MOST amino acids are degraded by transamination.
Enzyme: Aminotransferase (transaminase)
Coenzyme: PLP

General transamination reaction:

Amino acid + α-ketoglutarate ⇌ α-keto acid + glutamate

👉 Glutamate is the central “collection point” for amino groups.

Clinical tie-in:

• SGOT / AST (aspartate aminotransferase)

• SGPT / ALT (alanine aminotransferase)
  Elevated in liver or heart damage.

2. Removal of Ammonia & Transport to the Liver

Ammonia is toxic → must be safely moved to the liver as:

Two main carriers:

• Glutamine (blood-safe ammonia transporter)

• Alanine (via the glucose-alanine cycle from muscle)

Then ammonia is released in liver mitochondria for the urea cycle.

3. The Urea Cycle (Krebs–Henseleit Cycle)

Purpose: detoxify NH₃ → urea (non-toxic, excreted in urine).

You MUST know where the atoms of urea come from:

• One nitrogen → carbamoyl phosphate (from NH₃)

• One nitrogen → aspartate

• Carbon → CO₂ (as carbamoyl phosphate)

Regulator of the cycle:

• N-acetylglutamate (NAG) activates CPS-I

4. Carbon Skeleton Fates = Glucogenic vs Ketogenic

Fig. 18.12 summarizes this beautifully.

Glucogenic amino acids

→ enter TCA as pyruvate, OAA, α-KG, succinyl-CoA, fumarate
(Used to make glucose)

Ketogenic amino acids

→ acetyl-CoA or acetoacetate
(Used to make ketone bodies / fatty acids)

Only 2 purely ketogenic AA:

• Leucine

• Lysine

Ones that are BOTH:

• Isoleucine, Phenylalanine, Tyrosine, Tryptophan, Threonine

Essential AA

You cannot synthesize them, so they must come from diet.
Typically because their pathways are too long/complex/energy expensive.

The essentials (memory hack: "PVT TIM HALL"):

• Phenylalanine

• Valine

• Tryptophan

• Threonine

• Isoleucine

• Methionine

• Histidine

• Arginine* (essential in kids)

• Leucine

• Lysine

Nonessential AA

You CAN make these from basic metabolic intermediates:

• Alanine

• Aspartate

• Asparagine

• Glutamate

• Glutamine

• Glycine

• Serine

• Proline

• Cysteine

• Tyrosine*

(*Tyrosine is nonessential as long as you have phenylalanine.)

Where do amino acids come from?” — Carbon Skeleton Origins

Slide shows this beautifully:
A.A. carbon skeletons come from three major pathway families:

🔹 1. Glycolysis intermediates

Examples:

• 3-phosphoglycerate → serine → glycine

• Pyruvate → alanine; valine; leucine; isoleucine

🔹 2. Pentose phosphate pathway intermediates

• Erythrose-4-phosphate → aromatic AAs (Phe, Tyr, Trp)

🔹 3. TCA cycle intermediates

• α-KG → glutamate → glutamine → proline, arginine

• OAA → aspartate → asparagine → methionine, threonine, lysine, isoleucine

Key Amino Acid Synthesis Pathways A. Synthesis of the “basic 5”: Ala, Asp, Glu, Asn, Gln

All formed by simple transamination or amide transfer.

• Alanine ← pyruvate

• Aspartate ← OAA

• Glutamate ← α-KG

• Asparagine ← Asp + Gln (asparagine synthase)

• Glutamine ← Glu + NH₃ (glutamine synthetase)

➡ These are the “starter amino acids” for building more complex ones

B. Serine & Glycine (from 3-phosphoglycerate)

Steps:

1. 3-PG → 3-phosphohydroxypyruvate

2. → 3-phosphoserine

3. → Serine
   Serine → Glycine (via serine hydroxymethyltransferase; uses THF)

➡ THF links one-carbon metabolism to amino acid synthesis.

C. Aromatic amino acids (Phe, Tyr, Trp)

These are synthesized via very long pathways in bacteria/plants.
Humans cannot do this → they are essential (except Tyr).

D. Branched-chain AA biosynthesis (Val, Leu, Ile)

Again, only bacteria/plants can do this — long pathways, essential in humans.

E. Other important derivatives (exam favorites)

• Methionine → S-adenosyl methionine (SAM)

  • Universal methyl donor

• Homocysteine metabolism

  • Defect → homocystinuria (mental retardation + vascular damage)

• Arginine → nitric oxide & creatine phosphate

• Tryptophan → serotonin

• Tyrosine → dopamine → norepinephrine → epinephrine

• Glutamate → GABA, glutathione