전영주 - Disposal of amino acids

Lecture Information

• Topic: Degradation and synthesis of Amino Acids (Metabolism, Chapters 19-21)

• Instructor: 전영주

Key Concepts

• Protein degradation: Breaking peptide bonds to oligopeptides, then to single free amino acids

• First step of amino acid catabolism: Removal of amino group (e.g., from aspartate nitrogen) → ammonia via urea cycle

• Remaining carbon skeleton (alpha-keto acid): Glucogenic or ketogenic; some convert to metabolic intermediates for non-essential amino acid synthesis

• Chapters covered: 19 (amino acid metabolism), 20, 21

• Amino acids serve as precursors for DNA/RNA (genetic information storage); to be covered next Wednesday

• Amino acid metabolism disorders: Caused by defects in catabolism steps (almost 100% of cases), leading to accumulation of neurotoxic metabolic intermediates that cause intellectual disability and developmental disorders (all 100% neurotoxic)

• Example: Glutamate is a key neurotransmitter amino acid used exclusively in the brain; generated via oxidative deamination

• Glutamate acts as a signaling molecule in brain neurotransmission pathways, highlighting importance of amino acid metabolism

• Phenylalanine degradation (continued from yesterday): Starts with (e.g., 유니스피스 옥시더티브 디아미네이션) hydroxylation (by phenylalanine hydroxylase enzyme, using tetrahydrobiopterin cofactor) to tyrosine

• Tyrosine then converted to p-hydroxyphenylpyruvate, fumarate, and acetoacetate

• Phenylalanine and tyrosine are both glucogenic and ketogenic

• Methionine degradation is important (forms succinyl-CoA)

• MAT (methionine adenosyltransferase): Enzyme uses gating loop (alpha helix) for conformational change to cover active site, preventing substrate release after binding; allows catalysis (e.g., total methylation); positions methionine near ATP in active site; promotes catalysis; alpha helix gating loop does not form active site but promotes catalysis and positions methionine close to ATP; ATP binds via structural changes to keep methionine from escaping

• Gating loop mechanism: Substrate binds → loop closes (covers site) → catalysis → product forms → loop opens → product releases → disordered state for next substrate; enzyme induces conformational change in gating loop and stabilizes substrate binding for tighter interaction; enzyme itself undergoes conformational change via gating and enhances substrate-substrate binding stability

• Protein crystallography (in vitro): Uses stable substrate mimics (e.g., for MAT, mesionic acid + ATP mimic, as ATP unstable and hydrolyzes phosphate per second) to stabilize active site for structure determination

• Protein function determined by structure; functional maintenance involves structural/conformational changes (conformational change)

• Branched-chain amino acids (BCAAs): Defined by 3+ carbon atoms branching from alpha carbon; examples: isoleucine, leucine, valine; primarily catabolized in muscle; essential amino acids; key for brain development; important for protein synthesis and signaling pathways (e.g., glucose metabolism)

• BCAA catabolism: Begins with transamination by branched-chain amino acid aminotransferase (BCAT), removing amino group from alpha carbon (accepted by glutamate); similar to general amino acid catabolism process; catalyzed by BCAA aminotransferase, key for glutamate formation

• BCAAs crucial for neurodevelopment and brain development; their catabolism important for glutamate formation (key neurotransmitter in neurotransmission)

• Next step: Oxidative decarboxylation removes COO- group, converting alpha-keto acid to acyl-CoA; this process is very important

• Branched-chain alpha-keto acid dehydrogenase (BCKDH): Massive enzyme complex with numerous subunits; evolutionarily key (worth investing in); mediates second step

• Next: Dehydrogenation by acyl-CoA dehydrogenase to form enoyl-CoA (removes hydrogen)

• Isoleucine, valine, leucine share same transamination, oxidative decarboxylation, dehydrogenation processes initially, then diverge to form respective acyl-CoAs (e.g., acetoacetate from isoleucine)

• BCKDH (second step enzyme, oxidative decarboxylation): Massive complex composed of multiple polypeptide subunits (each polypeptide = subunit); requires cofactors: thiamine pyrophosphate, FAD, NAD+, lipoamide, coenzyme A

• BCKDH complex composed of 3 enzymes (E1, E2, E3), each with multiple subunits: E1 (alpha-ketoacid dehydrogenase), E2 (dihydrolipoyl transacetylase), E3 (dihydrolipoyl dehydrogenase)

• BCKDH complex structure: E1 (numerous, with sub-type post-translational phosphatase; forms C-panel), E2 core (central, highly valued/protected), E3 enzyme

• E2 domains: mating domain (binds E1 and E3), lipoyl domain (highly mobile/fluid, substrate channeling)

• Oxidative decarboxylation of branched-chain alpha-keto acids (nitrogen already removed) by E1 subunit (alpha-ketoacid dehydrogenase): performs decarboxylation; decarboxylated acyl group transferred to E2's lipoamide cofactor

• Acyl group then transferred from lipoamide to coenzyme A, forming acyl-CoA

• Reduced lipoamide requires oxidation for next reaction, handled by E3 (dihydrolipoyl dehydrogenase), which uses FAD and NAD+ as electron acceptors to reoxidize it for the next reaction

• Remaining steps not necessary to review in detail (instructor said "no need to look at the processes below"): Leucine, isoleucine, valine form products (e.g., glutamate) via transamination

• BCAA alpha-keto acids (after transamination) undergo oxidative decarboxylation by massive BCKDH complex (E1, E2 core, E3, phosphatase), forming acyl-CoA

• Amino acid catabolism defects (almost 100% of cases): Caused by step-wise defects in amino acid catabolism, leading to accumulation of metabolic intermediates that cause intellectual disability and developmental disorders

• Example: Hyperphenylalaninemia (PKU) - direct cause is defect in phenylalanine hydroxylase; prevents catabolism of phenylalanine to tyrosine (via hydroxylation using tetrahydrobiopterin cofactor), leading to phenylalanine accumulation and tyrosine deficiency

• PKU caused by phenylalanine hydroxylase defect (almost all from missense mutations); missense mutations change protein characteristics

• Missense mutations result in increased protein degradation rate via ubiquitin-proteasome system (UPS): amino acid substitution leads to misfolding → rapid degradation by UPS or formation of oligomisfolded aggregates (these are PAH enzyme defects)

• Not primarily due to loss of enzymatic function, but accelerated degradation of misfolded protein (missense mutation increases degradation rate rather than causing functional defect)

• PKU results in elevated phenylalanine, elevated phenylketones (phenylketonuria), decreased tyrosine levels

• Therapeutic strategies: Chaperoning to refold misfolded proteins or inhibiting degradation

• Tyrosine deficiency (tyrosine is substrate for neurotransmitters, to be covered soon) causes neurotransmitter synthesis issues, leading to various diseases

• PKU classification by plasma phenylalanine levels:

  • Classical PKU: ≥1200 μmol/L

  • Atypical/mild PKU: 600-1200 μmol/L

  • Mild hyperphenylalaninemia: 120-600 μmol/L

• Additional causes: Defects in tetrahydrobiopterin synthesis enzyme or in its regeneration from dihydrobiopterin (via reductase); results in hyperphenylalaninemia due to inability to metabolize phenylalanine

• Overall, defects in phenylalanine hydroxylase pathway lead to phenylalanine accumulation and hyperphenylalaninemia

• Phenylalanine hydroxylase (PAH): Composed of 3 functional domains; forms tetramer (4 subunits) required for activity

• N-terminal domain 1: Regulatory domain for allosteric activation by phenylalanine (cooperative, sigmoidal binding); composed of alpha helices

• Central domain: Catalytic core for conversion to tyrosine

• C-terminal domain: Multimerization domain (alpha helices bind 4 subunits together for activation)

• N-terminal regulatory domain shows cooperative, sigmoidal binding similar to hemoglobin's tetrameric cooperative regulation (GTLG: one subunit binding increases affinity in others by hundreds-fold); allosteric regulation by substrate phenylalanine (binding at allosteric site outside active site; one monomer's phenylalanine binding increases affinity in other monomers' active sites, boosting reaction rate)

Important Points

• Homocystinuria: Caused by defect in cystathionine beta-synthase (first step in methionine metabolism pathway: homocysteine + serine → cystathionine → cysteine + alpha-ketobutyrate); results in elevated homocysteine (in plasma/urine) and methionine levels, decreased cysteine; symptoms include lens dislocation, intellectual disability, thromboembolism

• Maple Syrup Urine Disease (MSUD): Caused by defect in branched-chain alpha-keto acid dehydrogenase (BCKDH, key enzyme for oxidative decarboxylation in BCAA catabolism); affects immune system, musculoskeletal system, central nervous system; leads to developmental disorders, neurological disorders, brain damage; characterized by maple syrup smell in urine

• MSUD treatment: Branched-chain amino acid (BCAA) restriction diet; early diagnosis and lifelong diet therapy can largely prevent neurological/developmental disorders; without early diagnosis, can lead to death within a few weeks after birth

• Importance of BCAA catabolism in brain: Generates glutamate, a key CNS neurotransmitter essential for brain development, cognition, memory, and learning; failure to form glutamate causes various diseases

• Leucine (among BCAAs): If not metabolized and accumulates, directly neurotoxic; plays key role in water homeostasis (when water supply disrupted, leucine critical for water homeostasis maintenance); if levels not properly maintained, water enters cells causing swelling

• Leucine catabolism intermediate (alpha-ketoisocaproic acid): Accumulates if not converted to final products; itself neurotoxic, causing encephalopathy (known to cause brain encephalopathy; acts as single agent)

• Treatment approach for hyperphenylalaninemia: Understanding PAH domain structure for targeted interventions (e.g., domains for allosteric activation, catalysis, multimerization)

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• Next class: Continue tomorrow at 11 AM

Questions & Discussion

• Student question: Why remember GTLG? (in context of cooperative regulation discussion, referencing hemoglobin's tetrameric cooperative sigmoidal binding where oxygen binding to one subunit increases affinity in others by hundreds-fold)

• Instructor explanation: Allosteric regulation (binding at site outside active site); phenylalanine binding to one monomer increases binding affinity in adjacent monomers' active sites (cooperative sigmoidal, like hemoglobin where O2 binding to one subunit increases affinity in others by hundreds-fold), boosting reaction rate

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Additional Learning

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