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Amino Acids: Disposal of Nitrogen

Chapter 19 - Lippincott's Illustrated Reviews: Biochemistry

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I. Overview of Amino Acid Catabolism

• Phase 1 - Nitrogen Removal: The α-amino group is removed, generating free ammonia (NH₃) and an α-keto acid. Ammonia is highly toxic, especially to the CNS, and must be quickly converted to a safe form - primarily urea - for excretion.
• Phase 2 - Carbon Skeleton Fate: The remaining carbon skeletons (α-keto acids) enter central metabolic pathways and can be converted to CO₂ (via TCA), glucose (gluconeogenesis), lipids, or ketone bodies.

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II. Nitrogen Metabolism & the Amino Acid Pool

A. The Amino Acid Pool

• Digestion and absorption of dietary protein
• Breakdown of endogenous (body) proteins
• Synthesis of nonessential amino acids

• Protein synthesis (structural, enzymatic, hormonal proteins)
• Biosynthesis of nitrogen-containing compounds (purines, pyrimidines, heme, creatine, etc.)
• Energy production - amino acids oxidized to CO₂, converted to glucose or fats

B. Protein Turnover

• Regulatory proteins (enzymes, transcription factors): short half-life (minutes to hours)
• Structural proteins (collagen, muscle fibers): long half-life (days to years)

C. Protein Degradation Pathways

• Proteins tagged with multiple ubiquitin molecules
• Tagged protein fed into the proteasome barrel
• Cleaved into amino acid fragments that are recycled
• Highly selective - the N-terminal amino acid influences the half-life (N-end rule)
• Proteins with PEST sequences (Pro, Glu, Ser, Thr-rich) are rapidly degraded

• Degrades extracellular and membrane proteins via acid hydrolases (cathepsins)
• Autophagy channels cytoplasmic contents into lysosomes

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III. Digestion of Dietary Proteins

Overview

• ~70-100 g of protein are consumed daily
• Proteins must be broken down to di-/tripeptides and free amino acids for absorption
• Digestion involves coordinated action of stomach, pancreas, and small intestine enzymes

A. Gastric Digestion

• HCl (secreted by parietal cells):
  • Denatures/unfolds proteins, exposing peptide bonds
  • Converts inactive pepsinogen → active pepsin (also autocatalytic at low pH)
• Pepsin (secreted by chief cells):
  • Active only at low pH (~1-2)
  • An endopeptidase - cleaves internal peptide bonds, producing polypeptides and a few free amino acids
  • Preferentially cleaves next to aromatic/large hydrophobic residues

B. Pancreatic Digestion

• Endopeptidases: Trypsin, chymotrypsin, elastase (cleave internal bonds at specific residues)
• Exopeptidases: Carboxypeptidases A and B (cleave from the C-terminus)

1. Enteropeptidase (brush border enzyme of duodenum) cleaves trypsinogen → trypsin
2. Trypsin then activates ALL other zymogens (amplification cascade)
3. Hormonal control: Secretin stimulates bicarbonate release; CCK (cholecystokinin) triggers enzyme and bile release

• Cystic Fibrosis / Pancreatitis: Impaired pancreatic secretion → incomplete digestion → steatorrhea and protein malnutrition
• Celiac Disease: Gluten-induced immune damage to intestinal villi → malabsorption

C. Small Intestine Brush Border

• Aminopeptidase (exopeptidase): cleaves amino acids from the N-terminus of oligopeptides, releasing free AAs and smaller peptides

D. Absorption

• Free amino acids: Absorbed via Na⁺-coupled co-transport into enterocytes (secondary active transport)
• Di- and tripeptides: Absorbed via H⁺-coupled transport (PepT1 transporter), then hydrolyzed by intracellular peptidases
• Only free amino acids enter the portal circulation to reach the liver
• Exception: Branched-chain amino acids (Val, Leu, Ile) largely bypass the liver and are taken up directly by muscle

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IV. Transport of Amino Acids into Cells

• Intracellular amino acid concentration is maintained higher than extracellular via active transport (ATP-dependent)
• At least 7 transport systems exist with overlapping specificities
• Shared transport systems are present in the small intestine AND renal proximal tubule

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V. Removal of Nitrogen from Amino Acids

A. Transamination - The Nitrogen Funnel

• Most amino acids donate their α-amino group to α-ketoglutarate
• Products: Glutamate + corresponding α-keto acid
• Catalyzed by aminotransferases (transaminases)
• Glutamate becomes the central nitrogen collector of the body

B. Mechanism of Aminotransferases

• PLP is covalently bound to a lysine residue at the active site (Schiff base)
• Step 1: Amino group transfers from amino acid → PLP → forms pyridoxamine phosphate (PMP) + α-keto acid
• Step 2: PMP donates amino group to a new α-keto acid → new amino acid + regenerates PLP

• Normally these enzymes are intracellular; only tiny amounts appear in blood from routine cell turnover
• Elevated plasma ALT/AST = tissue damage/necrosis

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VI. Oxidative Deamination

Glutamate Dehydrogenase (GDH)

• Location: Liver and kidney mitochondria
• Reaction: Glutamate + NAD⁺ (or NADP⁺) → α-ketoglutarate + NH₃ + NADH (or NADPH)
• Releases free NH₃ for urea synthesis
• Provides α-ketoglutarate for TCA cycle entry

• NAD⁺ → oxidative deamination (catabolism)
• NADPH → reductive amination (anabolism)

• GTP inhibits (energy-rich state → suppress catabolism)
• ADP activates (energy-poor state → increase amino acid degradation)

D-Amino Acid Oxidase (DAO)

• FAD-dependent, located in peroxisomes
• Metabolizes dietary D-amino acids (non-proteinogenic, mirror images of L-amino acids)
• Products: α-keto acids + NH₃ + H₂O₂ (peroxide broken down by catalase)
• Also degrades D-serine - linked to NMDA receptor modulation; altered DAO activity is implicated in schizophrenia risk

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VII. Ammonia Transport to the Liver

1. Glutamine Transport (Most Tissues - brain, muscle, liver)

• Glutamine synthetase (requires ATP): NH₃ + Glutamate → Glutamine (non-toxic, neutral)
• Glutamine travels in the blood to the liver and kidneys
• Glutaminase in the liver: Glutamine → Glutamate + NH₃ (released for urea synthesis)
• In kidneys: NH₄⁺ is excreted to regulate acid-base balance

2. Alanine Transport - The Glucose-Alanine Cycle (Muscle-specific)

• In muscle: Pyruvate + Glutamate → Alanine (via ALT) - packages nitrogen safely
• Alanine is transported to the liver
• In liver: Alanine → Pyruvate + Glutamate (via ALT, reversed)
• Pyruvate → glucose via gluconeogenesis → returns to muscle
• This cycle transfers nitrogen from muscle to liver while recycling carbon for energy

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VIII. The Urea Cycle

Overview

• Urea is the primary nitrogen disposal product in humans (~90% of urinary nitrogen)
• Each urea molecule contains 2 nitrogen atoms:
  • 1 from free NH₃ (released by GDH from glutamate)
  • 1 from aspartate (donated via AST transamination)
• Carbon and oxygen come from CO₂/HCO₃⁻
• Site: Primarily in hepatocytes (both mitochondria and cytoplasm)
• Excretion: Urea diffuses into blood → kidneys → urine

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Steps of the Urea Cycle

• Enzyme: Carbamoyl Phosphate Synthetase I (CPS I)
• Substrates: NH₃ + CO₂/HCO₃⁻ + 2 ATP
• Cofactor/Activator: N-acetylglutamate (NAG) - essential allosteric activator
• Location: Mitochondrial matrix
• This is the rate-limiting step of the urea cycle

• Enzyme: Ornithine Transcarbamylase (OTC)
• Carbamoyl phosphate + Ornithine → Citrulline + Pi
• Citrulline exits the mitochondria via ornithine/citrulline antiporter

• Enzyme: Argininosuccinate Synthetase
• Citrulline + Aspartate + ATP → Argininosuccinate + AMP + PPi
• This step incorporates the second nitrogen (from aspartate)
• Uses 1 ATP (but equivalent to 2 ATP because PPi is hydrolyzed)

• Enzyme: Argininosuccinate Lyase
• Argininosuccinate → Arginine + Fumarate
• Fumarate links the urea cycle to the TCA cycle (can be converted to malate → OAA → aspartate, regenerating the aspartate donor)

• Enzyme: Arginase I (virtually exclusive to liver)
• Arginine + H₂O → Urea + Ornithine
• Ornithine re-enters the mitochondria to repeat the cycle
• Arginase II in kidneys regulates arginine for nitric oxide synthesis (does NOT make urea)

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Stoichiometry

Aspartate + NH₃ + CO₂ + 3 ATP + H₂O →
    Urea + Fumarate + 2 ADP + AMP + 2 Pi + PPi

• 4 high-energy phosphate bonds consumed per urea molecule (3 ATP → 2 ADP + 1 AMP)
• Reaction is irreversible (large negative ΔG)

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Regulation of the Urea Cycle

• Key enzyme: CPS I (rate-limiting)
• N-acetylglutamate (NAG) is the essential allosteric activator of CPS I
  • NAG is synthesized by N-acetylglutamate synthase (NAGS) from Acetyl-CoA + Glutamate
  • Arginine activates NAGS → more NAG → more CPS I activity (positive feedback)
• Cycle rate increases after a protein-rich meal (more substrate, more arginine, more NAG)
• Long-term regulation: high-protein diet induces urea cycle enzyme synthesis

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Fate of Urea

• Diffuses from liver → bloodstream → kidneys → filtered and excreted in urine
• ~25% diffuses into the intestine → gut bacteria urease converts urea → CO₂ + NH₃
• NH₃ is partly lost in feces, partly reabsorbed into portal blood
• In renal failure: elevated plasma urea drives more urea into gut → more bacterial NH₃ production → worsening hyperammonemia
• Treatment: oral neomycin (antibiotic reduces gut bacteria → less NH₃ production)

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IX. Metabolism of Ammonia

Sources of Ammonia

Normal Ammonia Levels

• Normal plasma [NH₃]: 5-35 µmol/L
• Ammonia at 1000 µmol/L = medical emergency
• Predominantly transported as glutamine (non-toxic) and alanine

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X. Hyperammonemia

CNS Effects of Ammonia Toxicity

• Depletes α-ketoglutarate (TCA cycle intermediate) → impairs brain energy production
• Stimulates glutamine synthesis in astrocytes → astrocyte swelling → cerebral edema
• Inhibits neuronal function

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Types of Hyperammonemia

• Liver disease (hepatitis, cirrhosis): hepatocytes destroyed → impaired urea cycle
• Portal-systemic shunting: blood bypasses liver → ammonia escapes urea cycle → accumulates in systemic circulation

• Genetic deficiency of urea cycle enzymes
• OTC (Ornithine Transcarbamylase) deficiency: Most common urea cycle disorder; X-linked; presents in male newborns in first weeks of life with hyperammonemia, vomiting, lethargy
• All other urea cycle disorders follow autosomal recessive inheritance
• Arginase deficiency: Milder than other urea cycle defects
• Note: With urea cycle defects, arginine becomes an essential amino acid

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Treatment of Hyperammonemia

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Summary: Integration of Nitrogen Metabolism

Dietary Protein
    ↓ (digestion & absorption)
Amino Acid Pool
    ↓ (aminotransferases - PLP-dependent)
Glutamate (central nitrogen collector)
    ↓ (Glutamate Dehydrogenase)
NH₃ + α-KG
    ↓
Transport to liver (as Glutamine or Alanine)
    ↓
UREA CYCLE (liver mitochondria + cytoplasm)
    ↓
Urea → Kidney → Urine