Protein Translation and Amino Acid Metabolism Notes

Initiation Complex

• Composed of mRNA, tRNA, and the 50S ribosomal subunit.

• tRNA carries methionine (eukaryotes) or formal methionine (prokaryotes).

Aminoacylation

• Two processes depending on methionine/formal methionine placement:

  • Start codon.

  • Middle of the protein.

Initiation Factors

• IF1 and IF3.

• IF1 in the A site prevents tRNA alignment in the P site.

• IF3 in the E site prevents premature assembly of ribosomal subunits.

• IF2 (GTPase) recruits the start codon tRNA and cleaves GTP to release IFs and allow the large ribosomal subunit to bind.

Shine-Dalgarno Sequence

• A region ahead of the start codon complementary to the 16S rRNA in the small ribosomal subunit.

• Ensures start codon alignment in the P site.

• Varies depending on the organism; the start codon is relatively consistent.

Elongation Step

• Most of the protein synthesis pathway.

• Nucleophilic attack by the N-terminus of the amino acid in the A site on the carbonyl carbon of the amino acid in the P site.

• Elongation factor (EF-GTP) lands in the A site to move everything over one space akin pressing “enter” on a typewriter via GTP hydrolysis and causes a shift: P site tRNA moves to the E site and the A site tRNA moves to the P site.

Termination Step

• Release factor (RF) binds when a stop codon enters the A site.

• RF hydrolyzes the aminoacyl linkage between the peptide and tRNA.

• Ribosomal components dissociate and can be recycled or degraded.

Energy Cost

• Translation is energy-intensive, requiring about 4 ATP per amino acid addition.

• Energy is used in charging aminoacylated tRNA and EF-GTPase activity.

Amino Acid Oxidation

• Important for maintaining physiological metabolism.

• Influenced by diet: herbivores use <5% energy from amino acids, carnivores >90%.

• Can be upregulated during caloric deficit; however, not as efficient as glucose for energy storage.

• Evolutionarily significant to balance energy needs with protein degradation.

Protein Turnover

• Proteins can be broken down for energy when carbohydrates are scarce.

• Enzymes called peptidases break down peptides, often found in the small intestine.

• Peptidases can be specific for certain regions or amino acids in the peptide.

Pepsinogen and Pepsin

• Pepsin is stored in the zymogen form (pepsinogen) to prevent erroneous activation.

• Links cellular metabolism (nitrogen movement) with larger physiological metabolism (protein intake).

Amino Acid Metabolism

• Amino acids from diet or intracellular protein turnover are degraded.

• Separated into carbon skeletons and nitrogen.

• Carbon skeletons convert to alpha-keto acids.

• Nitrogen is processed via the urea cycle or used in biological synthesis.

Urea Cycle

• Occurs in organisms with a liver.

• Transamination reactions move nitrogen.

• Glutamate and glutamine are key amino acids in nitrogen processing.

• Alpha-ketoglutarate + nitrogen → glutamate.

• Glutamate + nitrogen → glutamine.

• For terrestrial vertebrates, urea is created to expel nitrogen.

• Formula: $(NH_4)$

• Urea has two nitrogens, requiring two ammonias. The equation for urea is: $(CO(NH<em>2)</em>2)$

Uric Acid

• Insoluble; excreted by some vertebrates like birds as a paste.

Enzymatic Transamination

• Movement of an amine with the help of an enzyme, often with pyridoxal phosphate cofactor.

• Alpha-ketoglutarate (α-KG) often accepts amino groups, generating glutamate.

• L-glutamine is a temporary nitrogen store; more prevalent in circulation.

• Equation: Alpha KG + nitrogen becomes glutamate.

• Then glutamate plus more nitrogen becomes glutamine.

Ammonia Removal

• Oxidative deamination reaction in the mitochondrial matrix generates reduced electron carriers (NADH or NADPH).

• This ammonia is then processed into urea for excretion.

Glutamine Synthetase

• Glutamine synthetase phosphorylates a molecule by expanding ATP.

• It can create enough local energy to allow for the addition of nitrogenous groups.

Alanine

• Muscle cells consume excess glucose under stress.

• Upregulate glycolysis, generating pyruvate.

• Pyruvate combines with nitrogen to form alanine.

• Alanine formula equation: $(C<em>3H</em>7NO_2)$

Alanine Transports

• Transports amino acid into the liver.

• Alanine goes through a transamination reaction to make glutamate.

• Glutamate can go through the urea cycle and regenerate pyruvate to go through the glucose-alanine cycle

Liver and Urea Cycle

• Located in the mitochondrial matrix of liver cells (hepatocytes).

• Goal: to get two nitrogens close to each other separated via a carbonyl bond.

• Has 3 sources that generate nitrogen. Those are glutamine from the blood, mystery pool of amino acids and alanine.

• Alanine comes from muscle cells. Alanine undergoes a transamination reaction in order to become glutamate.

Mitochondrial Matrix

• Glutaminase removes one nitrogen molecule from glutamine and regenerates glutamate.

• Two routes for glutamate that come from nitrogen:

  • Pathway 1 (blue) - into the free ammonia --> get converted into urea

  • Route 2 - nitrogen molecules onto oxaloacetate.

• This produces aspartate. This is new amindo acid formation - oxaloacetate + amino group is aspartate.

• Our overall aim is carbomoyl phosphate.