The Urea Cycle

Overview & Purpose

• Normal blood ammonia: 30–60 μM
• Urea accounts for ~90% of urinary nitrogen
• The cycle is almost exclusively hepatic

Compartmentalization

The 5 Reactions

Step 1 — Carbamoyl Phosphate Synthesis (Mitochondria)

NH₄⁺ + HCO₃⁻ + 2 ATP → Carbamoyl phosphate + 2 ADP + Pi

• Enzyme: Carbamoyl Phosphate Synthetase I (CPS I)
• Location: Mitochondrial matrix
• Requires cleavage of 2 ATP — energetically costly
• Absolute requirement for the allosteric activator N-acetylglutamate (NAG)
• This is the committed, rate-limiting step of the cycle
• (CPS II, in the cytosol, uses glutamine as N-source for pyrimidine synthesis — a separate enzyme)

Step 2 — Citrulline Synthesis (Mitochondria)

Carbamoyl phosphate + Ornithine → Citrulline + Pi

• Enzyme: Ornithine Transcarbamylase (OTC)
• Ornithine is the regenerating carrier molecule (analogous to oxaloacetate in the TCA cycle)
• Citrulline exits to the cytosol via the ornithine/citrulline antiporter

Step 3 — Argininosuccinate Synthesis (Cytosol)

Citrulline + Aspartate + ATP → Argininosuccinate + AMP + PPi

• Enzyme: Argininosuccinate Synthetase (ASS)
• Aspartate donates the second nitrogen atom of urea
• Cleavage of ATP → AMP + PPi (equivalent to 2 ATP used)
• Aspartate is generated by transamination of oxaloacetate by AST

Step 4 — Argininosuccinate Cleavage (Cytosol)

Argininosuccinate → Arginine + Fumarate

• Enzyme: Argininosuccinate Lyase (ASL)
• Fumarate is a key byproduct — it links the urea cycle with the TCA cycle (the "Krebs bi-cycle")
• Fumarate → malate → oxaloacetate → transaminated back to aspartate (recycling the nitrogen carrier)

Step 5 — Arginine Hydrolysis / Urea Release (Cytosol)

Arginine + H₂O → Urea + Ornithine

• Enzyme: Arginase I (virtually exclusive to the liver)
• Releases urea from arginine's guanidinium group
• Regenerates ornithine, which re-enters the mitochondria to restart the cycle

Overall Stoichiometry

Aspartate + NH₃ + HCO₃⁻ + 3 ATP + H₂O
    → Urea + Fumarate + 2 ADP + AMP + 2 Pi + PPi

• 4 high-energy phosphate bonds are consumed per urea molecule
• The reaction is irreversible (large negative ΔG)
• Both nitrogen atoms of urea ultimately come from glutamate: one via oxidative deamination (→ NH₃) and one via transamination of oxaloacetate (→ aspartate)

Regulation

Nitrogen Sources Summary

Link to TCA Cycle — The "Krebs Bi-Cycle"

Clinical Relevance

Sources: Basic Medical Biochemistry: A Clinical Approach, 6e, pp. 1321–1326 | Biochemistry, 8th ed (Lippincott Illustrated Reviews), pp. 712–715

Structure of Proteins

The Building Blocks — Amino Acids & Peptide Bonds

• Has partial double-bond character → it is rigid and planar (no free rotation around it)
• Is uncharged at physiological pH (the C=O and N-H groups are polar but not ionizable)
• The N-terminal (free amino end) is written to the left; the C-terminal (free carboxyl end) to the right
• A chain of ≥50 amino acids is called a polypeptide
• Average molecular weight of one amino acid: ~120 Da

Level 1 — Primary Structure

• Encoded directly by DNA
• The ultimate determinant of all higher-order structure and function
• The basis for protein identity — mutations here (e.g., sickle-cell disease) cause misfolding and disease

Level 2 — Secondary Structure

α-Helix

• A right-handed spiral with the side chains (R groups) pointing outward
• Stabilized by H-bonds: the C=O of each peptide bond to the N-H four residues ahead
• 3.6 amino acids per turn
• Proline disrupts the helix (its ring creates a rigid kink)
• Examples: keratin (hair, skin), large portions of myoglobin

β-Sheet (β-Pleated Sheet)

• Two or more fully extended β-strands aligned laterally, linked by perpendicular H-bonds between strands
• Strands can be antiparallel (N-termini alternate) or parallel (N-termini on same side)
• R groups extend alternately above and below the plane of the sheet
• Has a slight right-handed twist

β-Bends (β-Turns / Reverse Turns)

• 4-amino acid loops that reverse the direction of the polypeptide chain
• Allow the protein to form a compact, globular shape
• Often contain proline and glycine
• Stabilized by H-bonds between residue 1 and residue 4 of the turn
• Frequently located on the protein surface

Supersecondary Structures (Motifs)

• β-α-β motif (β strand, helix, β strand)
• β-meander (antiparallel β-strands connected by loops)
• β-barrel (β-strands folded into a barrel shape)
• Helix-loop-helix (found in DNA-binding transcription factors)

Level 3 — Tertiary Structure

• Arrangement of secondary elements and loops into domains (functional/structural units, usually >200 residues have ≥2 domains)
• Compact globular structure with hydrophobic core and hydrophilic surface

Four Stabilizing Interactions

Protein Folding & Chaperones

• Folding occurs within seconds to minutes via ordered, nonrandom pathways
• Driven by the hydrophobic effect: hydrophobic groups come together as water is expelled
• Molecular chaperones (Heat-Shock Proteins, HSPs) facilitate correct folding:
  • Hsp70: binds extended nascent chains, prevents premature folding
  • Hsp60: cage-like barrel where misfolded proteins refold in an isolated environment
• Without chaperones, many proteins misfold and aggregate

Protein Denaturation

Level 4 — Quaternary Structure

• Subunits held together by non-covalent interactions (H-bonds, ionic, hydrophobic)
• Subunits may be identical (homomeric) or different (heteromeric)
• Classic example: Hemoglobin — a tetramer (2α + 2β subunits) exhibiting cooperative O₂ binding
• Isoforms / Isoenzymes: proteins performing the same function but with different primary structures (tissue-specific expression)

Protein Misfolding & Disease

Functions of Proteins

Sources: Biochemistry, 8th ed (Lippincott Illustrated Reviews), pp. 51–83 | Henry's Clinical Diagnosis and Management by Laboratory Methods, pp. 321–323