Comprehensive Study Notes on Protein and Amino Acid Metabolism

Generalities of Protein Metabolism

Total Protein Mass: An average 70 kg healthy adult body contains approximately 11 kg of protein (out of an estimated 10 kg to 11 kg total).
Body Compartments (Brozek's Model for 70 kg adult):
- P (Body Mass): $70\,kg$
- MG (Fat Mass): $10\,kg$
- ECF (Extracellular Fluid): $20\,L$
- ICF (Intracellular Fluid): $25\,L$
- MIN (Minerals): $4\,kg$
- PROT (Proteins): $11\,kg$
Classification of Protides:
- Peptides: Sequences of amino acids linked by peptide (amide) bonds.
  - Oligopeptides: Dipeptides (e.g., Carnosine: Ala-His), Tripeptides (e.g., Glutathione: Glu-Cys-Gly).
  - Polypeptides (9-51 Amino Acids): ADH ( $9\,AA$ ), Oxytocin ( $9\,AA$ ), Glucagon ( $29\,AA$ ), Insulin ( $51\,AA$ ), ACTH ( $39\,AA$ ), Alpha-MSH, Calcitonin, Parathormone, Angiotensin, Kinine.
- Proteins:
  - Simple Proteins (Holoproteins):
    - Globular Soluble: Protamine, histone, albumin, prolamine, gluteline, globulin.
    - Fibrillar Soluble: Fibrinogen, myofibril.
    - Fibrillar Insoluble: Collagen, keratin, elastin, silk fibroin.
  - Complex Proteins (Heteroproteins): Lipoproteins, glycoproteins, phosphoproteins.
Essential vs. Non-Essential Amino Acids (AA):
- Humans synthesize proteins from 20 main amino acids plus selenocysteine (found in deiodase, selenoprotein P, and glutathione peroxidase).
- Essentials (must be dietary): Leucine, Isoleucine, Valine, Methionine, Phenylalanine, Tryptophan, Threonine, Lysine.
- Semi-Essential: Histidine and Arginine.
- Non-Essential: Glutamine, Glutamate, Aspartate, Asparagine, Cysteine, Proline, Glycine, Tyrosine, Serine, Alanine.
Biological Value of Proteins:
- High Biological Value: Animal proteins (egg, milk, cheese, meat) containing all indispensable AA.
- Low Biological Value: Cereal grains, legumes, vegetables, and fruits (lacking certain essential AA).
- Exception: Quinoa is a rare cereal containing all 8 essential amino acids.
Protein Functions:
- Structure: Collagen, keratin.
- Enzymatic: Almost all enzymes.
- Hormonal/Neuromediators: Insulin, glucagon.
- Motor: Actin, myosin.
- Transport: Albumin.
- Transduction: Receptors, G-proteins.
- Immunity: Cytokines.
- Genome Regulation: Transcription factors.

Protein Structure and Turnover

Protein Turnover: The perpetual renewal of proteins in the body.
Complexity: There are roughly 10,000 different proteins in a eukaryote.
Primary Structure: Specific AA sequence. Contains covalent peptide and disulfide bonds. No weak bonds. Mass ranges from dipeptides ( $200\,kD$ ) to enzyme complexes ( $10^6\,kD$ ). Average protein contains $20\,AA$ , $8\%\text{ Leucine}$ , and has $1\,g\text{ Nitrogen}$ per $6.25\,g\text{ protein}$ .
Secondary Structure: Local folding. Uses peptide and disulfide (covalent) bonds and weak hydrogen (H) bonds. Rotation occurs around peptide bonds. Includes $\alpha$ -helices (keratin) and $\beta$ -sheets.
Tertiary Structure: Spatial folding involving distant AA. Bonds include weak interactions (hydrophobic, hydrogen, electrovalent) and covalent bonds (peptide, disulfide bridges). Organization into functional domains.
Quaternary Structure: Interactions (weak and covalent disulfide bridges) between multiple protein subunits (e.g., Hemoglobin, proteasome). Not present in all proteins.

Digestion and Absorption of Proteins

Daily Intake/Flow:
- Dietary intake: $70-100\,g/day$ (typically $1-1.5\,g/kg$ ).
- Secreted gastrointestinal proteins: $50\,g/day$ (enzymes, mucus, debris).
- Total flow through portal vein: Roughly $150\,g/day$ .
- Splanchnic extraction (liver/viscera) removes $60-80\%$ of absorbed AA (oxidation, protein synthesis). Only $20\%$ (mostly branched-chain AA) reach general circulation.
Digestive Enzymes:
- Stomach: Pepsinogen is activated by hydrochloric acid ( $HCl$ ) and autocatalysis to Pepsin. Pepsin cleaves at $\text{NH}_2$ of aromatic AA or Leucine.
- Intestine/Pancreas:
  - Bicarbonate: Neutralizes gastric acid.
  - Trypsin: Activated by Enteropeptidase; cleaves at $COOH$ of Arg or Lys.
  - Chymotrypsin: Cleaves at $COOH$ of aromatic AA or Leu.
  - Elastase: Cleaves at $COOH$ of short-chain AA (Ala, Gly, Ser).
  - Exopeptidases: Carboxypeptidase A (C-terminal aromatic) and B (C-terminal basic).
  - Intestinal Proteases: Aminopeptidases (N-terminal) and di/tri-peptidases.
Absorption Mechanisms:
- Sodium-Amino Acid Transport System: Uses a symporter for one Sodium ( $Na^+$ ) ion and one amino acid. Driven by the $Na^+\text{-}K^+\text{ ATPase}$ pump (3 $Na^+$ out, 2 $K^+$ in). Consumes $25\%$ of cellular ATP (up to $70\%$ in neurons). Inhibited by ouabain.
- \gamma-Glutamyl Cycle: AA reacts with glutathione (\gamma-glutamyl-cysteinyl-glycine) to form a \gamma-glutamyl-amino acid and cysteinyl-glycine. The AA is transported across the membrane and released into the cytoplasm.

General Amino Acid Metabolism

Catabolism Overview: AA are not directly used for energy like glucose. Excess AA have their carbon skeletons converted into Krebs cycle intermediates, glucose (gluconeogenesis), or ketone bodies (ketogenesis).
Deamination Reactions:
- Direct Deamination: Mostly Serine (via Serine Dehydrogenase to Pyruvate + $NH_4^+$ ) and Threonine (via Threonine Dehydrogenase to Alpha-ketobutyrate + $NH_4^+$ ).
- Oxidative Deamination:
  - L\n-amino-acid\,oxidase: FMN-linked, low activity.
  - D\n-amino-acid\,oxidase: FAD-linked (liver/kidney), acts on non-natural AA.
  - L\n-glutamate\,dehydrogenase: Abundant in liver/brain.
  - Reaction: Glu + H_2O + NAD(P)^+ \leftleftharpoons \alpha\text{-ketoglutarate} + NH_3 + NADH(P) + H^+
Transamination: Transfer of \alpha\n-amino groups to a ketoacid, catalyzed by aminotransferases (transaminases) requiring Pyridoxal Phosphate (PLP) as a coenzyme.
- Examples:
  - ASAT (Aspartate Aminotransferase/GOT): $Asp + \alpha\text{-ketoglutarate} \rightleftharpoons Glu + Oxaloacetate$ . High levels in muscle and liver ( $N < 14\,U/L$ ).
  - ALAT (Alanine Aminotransferase/GPT): $Ala + \alpha\text{-ketoglutarate} \rightleftharpoons Glu + Pyruvate$ . Specific to liver ( $N = 5\,to\,23\,U/L$ ).

The Urea Cycle (Ureogenesis)

Location: Occurs exclusively in the liver. Five reactions total (2 in mitochondria, 3 in cytosol).
Sequence of Reactions:
1. Carbamoyl Phosphate Synthesis: $CO_2 + NH_4^+ + 2\,ATP \rightarrow \text{Carbamoyl phosphate} + 2\,ADP + Pi$ . Enzyme: CPS I (requires N-acetyl glutamate).
2. Citrulline Formation: Carbamoyl phosphate + Ornithine $\rightarrow$ Citrulline. Enzyme: OTC (Ornithine Transcarbamoylase).
3. Argininosuccinate Synthesis: Citrulline + Aspartate + ATP $\rightarrow$ Argininosuccinate + AMP + PPi. Enzyme: Argininosuccinate synthetase.
4. Cleavage: Argininosuccinate $\rightarrow$ Arginine + Fumarate. Enzyme: Argininosuccinate lyase.
5. Hydrolysis: Arginine + $H_2O$ $\rightarrow$ Urea + Ornithine. Enzyme: Arginase.
Summary Equation: $NH_3 + CO_2 + \text{Aspartate} + 3\,ATP \rightarrow \text{Urea} + \text{Fumarate} + 2\,ADP + AMP + 2\,Pi + PPi$ .
Energy Balance: Uses 4 high-energy bonds ( $4 \times ATP$ ). However, the regeneration of Aspartate via Fumarate $\rightarrow$ Malate $\rightarrow$ Oxaloacetate produces 1 $NADH$ , equivalent to $3\,ATP$ , resulting in a net cost of only 1 phosphate bond.
Clinical Relevance:
- Hyperammonemia: Elevated ammonia causes brain damage, coma, and death due to excessive glutamate/glutamine levels depleting $\alpha\text{-ketoglutarate}$ .
- Deficiencies: Acquired (cirrhosis, alcoholism) or hereditary (leading to mental retardation).

Synthesis and Degradation of Specific Amino Acids

Synthesis Highlights:
- Tyrosine: Synthesized by Phenylalanine Hydroxylase from Phenylalanine. Requires $O_2$ , $Fe^{2+}$ , and Tetrahydrobiopterin ( $H_4BPt$ ). Deficiency causes Phenylketonuria (PKU).
- Alanine: From Pyruvate via transamination.
- Cysteine: From Methionine via transsulfuration.
- Arginine: Formed in the urea cycle.
Degradation Classification:
- Glucogenic (form pyruvate/Krebs intermediates): Asn, Asp (to Oxaloacetate); Arg, Glu, Gln, Pro, His (to $\alpha\text{-ketoglutarate}$ ); Ile, Val, Met (to Succinyl CoA via Propionyl CoA).
- Ketogenic (form Acetoacetate/Acetyl CoA): Leucine and Lysine (strictly ketogenic).
- Both: Phe, Tyr, Trp, Thr, Ile.
Branched-Chain AA (Val, Ile, Leu) Degradation:
- Requires transamination followed by oxidative decarboxylation by $\alpha\text{-keto acid dehydrogenase}$ (needs TPP, Lipoic acid, CoA, FAD, NAD).
- Deficiency leads to Maple Syrup Urine Disease.

One-Carbon Metabolism: Folate and Vitamin B12

Vitamin B9 (Folate/THF):
- Structure: Pterine + Para-aminobenzoic acid + Glutamate.
- Conversion: Folate $\rightarrow$ Dihydrofolate (FH2) $\rightarrow$ Tetrahydrofolate (FH4) via Folate Reductase.
- Derivatives: $N^{10}\text{-formyl-FH4}$ (Purine synthesis C2), $N^5, N^{10}\text{-methenyl-FH4}$ (Purine synthesis C8), $N^5, N^{10}\text{-methylene-FH4}$ (dUMP to dTMP), $N^5\text{-methyl-FH4}$ (transfer to B12).
Vitamin B12 (Cobalamin):
- Source: Animal origin (meat, fish, milk). Requires intrinsic factor for absorption.
- Cofactor Roles:
  - Methylmalonyl CoA Mutase: Rearrangement of methylmalonyl-CoA to succinyl-CoA.
  - Methionine Synthase: Transfer of methyl group from FH4 to homocysteine to form Methionine.
- Deficiency Symptoms: Weakness, numbness (myelin maintenance), depression, dementia, anemia (bone marrow regeneration).

Other Amino Acid Derived Products

Creatine: Produced in the liver from Glycine, Arginine, and SAM. In muscles/brain, stored as Creatine Phosphate (by CK/CPK). Non-enzymatically cycles into Creatinine for renal excretion.
Decarboxylation Products:
- GABA: From Glutamate (inhibitory neurotransmitter).
- Histamine: From Histidine (vasodilation, gastric acid stimulation).
- Putrescine/Spermine: From Ornithine.
- Taurine: From Cysteine.
- Serotonin/Melatonin: From Tryptophan.
- Nitric Oxide (NO): From L-Arginine via NO Synthase. Role: Vasodilator, neurotransmitter, microbicide.
Nucleotides:
- Purine Synthesis: Requires Asp, Gly, Gln, $CO_2$ , and Formyl-FH4. Degraded to Uric Acid.
- Pyrimidine Synthesis: Requires Asp, Gln, and $CO_2$ . Degraded to $\alpha\text{-ketoglutarate}$ , ammonia, and malonate.

Hemoglobin Metabolism

Synthesis (Heme):
1. $Succinyl CoA + Glycine \rightarrow \delta\text{-ALA}$ (Enzyme: ALA Synthase, coenzyme PLP).
2. 2 $\delta\text{-ALA}$ condense into Porphobilinogène (PBG).
3. 4 PBG condense to Uroporphyrinogen III.
4. Modification to Protoporphyrin IX.
5. Iron ( $Fe^{2+}$ ) insertion via Ferrochelatase.
Iron Metabolism:
- Absorption: Heme iron ( $20-30\%$ ) vs. non-heme iron ( $2-5\%$ ). Absorption enhanced by Vitamin C and inhibited by tannins (tea/coffee).
- Storage: Ferritin (liver/spleen) or Hemosiderin (excess).
- Transport: Transferrin.
Degradation: Red blood cells live 120 days. Phagocytosed in the SRE (spleen/liver).
- Heme $\rightarrow$ Biliverdin (green) + Iron + CO (Enzyme: Heme oxygenase).
- Biliverdin $\rightarrow$ Bilirubin (Enzyme: Biliverdin reductase).
- Bilirubin Transport: Bound to albumin in plasma.
- Liver Conjugation: Bilirubin + UDP-glucuronate $\rightarrow$ Bilirubin diglucuronide (soluble).
- Intestinal Conversion: To Urobiline and Stercobiline (brown color of feces).
Pathology (Jaundice/Ictère): Plasmatic Bilirubin $> 70\,\mu mol/l$ . Normal Total Bilirubin is $< 17\,\mu mol/L$ .

Thyroid Hormones

Production: Occurs in the thyroid follicle.
- Thyroglobulin (Tg): Dimeric protein ( $660\,kDa$ ) with 120 Tyrosine residues.
- Iodine Transport: $Na^+/I^-$ symporter; exits into colloid via Pendrin (antiport $Cl^-/I^-$ ).
- Mechanism: Oxidation of $I^-$ to $I_2$ by TPO. Iodation of Tyrosine leads to MIT and DIT.
  - $DIT + DIT \rightarrow T4$
  - $MIT + DIT \rightarrow T3$
Functions: Increases basal metabolism, heart rate (positive chronotropic), growth (myelination, linear bone growth).
Transport: Bound to TBG and transthyretin. Half-life: T4 ( $6.5\,days$ ), T3 ( $2.5\,days$ ). Only free hormones (T4L, T3L) are active.
Deiodination: T4 (prohormone) converted to T3 (active) by 5'-deiodase (Type 1 and 2). Type 3 inactivates them.