Amino Acid Pool and Nitrogen Metabolism

Amino Acid Pool and Nitrogen Metabolism

• Amino acids are derived from the amino acid pool
• Focus on the sources of amino acids
• Learn about protein digestion in the gastrointestinal tract
• Study intracellular digestion with Ubiquitin and Proteosomes
• Focus on intercellular digestion via lysosomes

The Amino Acid Pool

• Consists of body protein, dietary protein, liver synthesis and nitrogen compounds
• Amino acid pool is affected by digestion, catabolism and synthesis
• The pool drives urea and citric acid cycles and production of hormones and nucleotides

Protein and Amino Acid Catabolism: Digestion

• Proteins and big peptides require degradation into free amino acids before absorption in the gastrointestinal tract.
• Protein digestion begins in the stomach and continues in the small intestine.
• Gastric, pancreatic, and intestinal juices all facilitate digestion.

Digestive Enzymes: Gastric Juice

• Gastric juice contains HCl pH 2, produced by parietal cells, that eliminates microorganisms, denatures proteins, and creates an acidic environment that optimizes pepsin activity.
• Pepsin, secreted as pepsinogen, works optimally in acidic environments.
• Pepsinogen is autocatalytically converted to pepsin through the removal of a 44 amino acid N-terminal pepsin, hydrolyzing proteins into peptides and amino acids

Pepsin

• Is expressed as zymogen called pepsinogen, whose primary structure as an additional 44 amino acids
• In the stomach, chief cells release pepsinogen

Pepsinogen Activation

• Hydrochloric acid creates an acidic environment, allowing pepsinogen to unfold and cleave itself through an intramolecular reaction
• Reaction generates pepsin (active form) plus a 44 amino acid N-terminal fragment
• Pepsin cleaves 44 amino acids from pepsinogen molecules in an autocatalytic intermolecular reaction further generating pepsin.

Intramolecular Reaction

• Pepsin is involved in an intermolecular reaction
• In the intramolecular reaction, low pH initiates activation

Pancreatic Juice & Intestinal Juice

• Pancreatic juice has proteases secreted as proenzymes from Pancreatic Acinar cells.
• Enterokinase secreted by duodenal epithelial cells activates trypsinogen to trypsin by removing 6 amino acids.
• Trypsin activates proenzymes: chymotrypsin, elastase, carboxypeptidase A and B and the pancreas secretes these agents.
• Intestinal Proteases such as Aminopeptidase and Dipeptidase contribute to the digestion into free amino acids.
• Di- and Tri-peptides are usually absorbed and digested to free amino acids within the intestinal epithelial cells.

Action of the digestive proteases

• Pepsin, trypsin, chymotrypsin, and elastase are endopeptidases that hydrolyze peptide bonds
• Exopeptidases ; aminopeptidases remove the amino acid at the N-terminus and the carboxypeptidases remove the amino acid at the C terminus

Absorption of Amino Acids (AA)

• Free amino acid absorption occurs within the small intestine's lumen
• Amino acids are absorbed via secondary active Na+ dependent transport, facilitated diffusion, and the y-glutamyl cycle
• Gut lumen transports amino acids using 5 to 6 major transport mechanisms from the blood to the liver, muscle, and kidneys.

Transepithelial amino acid transport

• Na-dependent carriers transport both Na and an amino acid into the intestinal epithelial cell from the intestinal lumen
• Na is pumped out on the serosal side by the Na,K-ATPase in exchange for K.
• An amino acid is carried by a facilitated transporter down its concentration gradient into the blood, on the serosal side and this process represents secondary active transport.

Amino acid absorption via y-Glutamyl Cycle

• Amino acids get transported across the membrane by reacting with glutathione (y-glutamyl cysteinyl -glycine) to form y-glutamyl-aminoacid.
• An amino acid is released, and glutathione is resynthesized from cysteinyl-glycine and oxoproline in cells of intestine and kidneys.

Disorders associated with AA Transport

• Defects in AA transport lead to increased AA levels in urine (Amino Acidurias)
• Five major transport mechanisms are known for different amino acid groups
• Small amino acid specificity goes undetected
• Hartnup's Disease: Neutral and aromatic AA
• Cystinuria: BAsic AA and cystine.
• Glycinuria: Imino acids and glycine

Hartnup Disease

• Hartnup Disease is due to a defect in the transport mechanism for large neutral and aromatic AA
• Symptoms suffered are like in Pellagra.
• Niacin is deficient in patients which is synthesised from Tryptophan.
• Niacin is used to treat related symptoms.

Cystinuria & Glycinuria

• Cystinuria is a defect in the transport of basic amino acids, transporting cystine.
• Cystine: two cysteines joined by S-S bonds, relatively insoluble in urine.
• Cystine crystal deposits, urinary tract infections, and renal stones may occur as symptoms.
• Penicillamine , a drug, can overcome deposition of crystal, reacting with cystine to form water-soluble cysteine-penicillamine.
• Glycinuria is a defect in glycine, proline, and hydroxyproline transport, leading to their excretion in the urine with no clinical symptoms

Intracellular Protein Degradation

• Body proteins degrade into free AAs, resynthesized at various rates.
• Liver enzymes: Hours life while hemoglobin has 120 days half life
• Denatured proteins degrade rapidly.
• Lysosome activation causes more proteolysis.
• Glucocorticoids boost muscle protein proteolysis.
• Excess thyroid hormones elevate protein turnover.
• Insulin increases protein production and reduces proteolysis

Lysosomal pathways for protein degradation

• Cytoplasmic proteins and damaged organelles are degraded by lysosome through autophagy (autophagy-lysosomal pathway)
• Extracellular proteins are degraded by lysosome through endocytosis (endosome-lysosomal pathway)

Ubiquitin and its role in Degradation

• Protein degradation removes abnormal, defective, non-functional proteins that obstruct functional protein processes.
• Inducible enzymes are disposed if their activities become unnecessary.
• Ubiquitin protein (8.5kD, 76 amino acids) binds to proteins through its C-terminal glycine, forming a covalent bond with lysine residues. This is followed by degradation via ATP-dependent Proteosome S26.

Ubiqitinylation

• Targets proteins for degradation
• E₁= Ubiquitin activating enzyme
• E₂= Ubiquitin carrier protein
• E3= Ubiquitin ligase

Mechanism of Ubiquitinylation

• The E₁ enzyme first catalyzes Ubiquitin-E₁ complex with thioester link to SH group in E₁ with ATP
• The E₂ enzyme receives Ubiquitin from Ubiquitin-E₁ and releases E₁SH, then transfers Ubiquitin to protein via Enzyme3
• The E3 enzyme couples Ubiquitin molecule to protein tagged for degradation, releasing E₂SH.
• Usually several Ubiquitin molecules are placed on the degradable protein

Protein degradation

• Ub enzyme, E₁, carrier, and E3 ligands link Substrates to Ub molecule chains.
• In the 26S proteasome, the Ub-conjugate binds to the 19S regulatory component , the Ub chain is disassembled, by 20S core particle in an ATP linked process., and other enzymes hydrolyze peptides.

The role of the 26S Proteasome

• in the Pathogenesis of Human Diseases
• Deregulation of the ubiquitin/proteasome system contributes to various human diseases like cancer, neurodegenerative, and myodegenerative conditions.
• Cancer: deregulated proteasomal proteolysis of tumor suppressors/oncoproteins is associated with initiation, maintenance of neoplastic proliferation, tumor progression, and prognosis.
• Parkinson's Disease: selective cell death in dopaminergic neurons in substantia nigra
• Alzeimer's Disease: degeneration/loss of cortical/limbic neurons, and extracellular amyloid
• Polyglutamine and Muscle Dystrophy: expansion of CAG repeats in causative genes, resulting in polyglutamine stretches in encoded proteins.

Catabolism of Proteins and Amino acids

• Continuous protein synthesis and degradation is called Protein Turnover.
• Important in all life forms, a man converts 1-2% of the body protein, particularly muscle protein, each day.
• 75 - 80% of amino acids are re-utilized for protein production as urea while the remaining 20 - 25% get excreted.
• Carbon chains of amino acids can be degraded into amphibolic intermediates.

Proteins are Degraded at Varying Rates

• The degradation rate relies on the physiological demand.
• Its half-life, is the time protein concentration is reduced by 50%.
• Liver proteins have half-lives from 30 minutes to 150+ hours.
• Proteins having short half lives have the high amino acids PEST (Proline, Glutamate, Serine, and Threonine) sequences target breakdown.

Detection of Muscle Wasting

• Rapid degradation of muscle protein occurs in muscular dystrophy.
• 3-methyl histidine: indication of an amino acid marker as the histidine residues of actinomyosin get methylated post incorporation.
• When actinomyosin degrades, the 3-methyl histidine is released and excreted into the urine.
• Determination of 3-Methyl histidine amount in the urine signifies muscle breakdown.

Nitrogen Balance

• Nitrogen balance occurs when nitrogen consumption matches nitrogen loss (urine, skin, feces)
• Positive nitrogen, higher intake than output, occurs in healthy adults/children/recovering patients
• Negative nitrogen, when intake is lower than output, happens in disease/starvation and can become fatal during long periods.

Amino Acid Pool

• Dietary amino acids from what's left of protein digestion is a source for the AA
• Can be created from endogenous protein breakdown with Ubiquitin Proteosome S26
• Can be made from lysosomal degradation of endocytosed proteins.
• Nonessential amino acids can be synthesized
• AAs Arg, Phe, Thr, Trp, Lys, Thr, Ile, Leu, His, Val & Met are essential