Post midterm 2

• Amino acids from degraded proteins/diet can be used to form new proteins but …

  • they can also be used to support the formation of glucose during starvation or be used as a source of energy
  • Before entering the TCA cycle/ketogenesis, amino acids needs to be altered
  • The first step is most often the removal of the α- amino group of the amino acid
• With few exceptions, the first step in amino acid catabolism involves the removal of a- amino group to give the corresponding a- keto acid
• Amino acid catabolism: Overall scheme

  A) Amino acids

  B) a-keto acids

  C) NH4

  D) glutamate

  E) a-ketoglutarate

  F) NH4

  G) urea

  
• • An aminotransferase/transaminase is an enzyme that catalyzes a reaction between a keto acid and an amino acid
  • An amino group and = O are exchanged in this reaction

  
• • Glutamate plays a central role in collecting amino groups from various reactions
  • Amino acids travel to the liver

  
• Transamination

  • Transaminases are bound to the PLP coenzyme
  • First stage: transfer α-amino group onto transaminase, amino group is carried by coenzyme PLP ; PLP-dependent enzyme-catalyzed transamination
  • Second stage: transfer amino group from PLP onto a different α-keto acid, α- ketoglutarate is the major NH3 acceptor, glutamate is the major product
• Summary of transamination

  • PLP-dependent transamination reactions are reversible
  • Any amino acids can be used as substrate for the first stage (forward) reaction
  • α- ketoglutarate is the major α-keto acid substrate in the second stage (reverse) reaction
  • This reaction happens where the protein is degraded or when proteins are ingested/digested and reach the liver
• Transport of ammonia to the liver

  • Ammonia (NH3) generated from multiple processes, (i.e. protein degradation) can be found in peripheral tissues
  • Ammonia accumulation has toxic consequences
  • Liver is in charge of converting ammonia into urea
  • Ammonia is transported in the blood in the form of glutamine or alanine
• Transport of ammonia to the liver from:

  • amino acids - Ingested proteins/cellular proteins
  • alanine - muscle
  • glutamine - most tissues excluding muscle
• Production of alanine by the muscle

  • Transaminase reaction using pyruvate generated by glycolysis as a terminal transamination substrate
  • Reaction is catalyzed by alanine transaminase
  • In the process, α- ketoglutarate is recycled and alanine is generated
  • Using the bloodstream, alanine will be transported to the liver where it will be converted back to pyruvate
  • The reversal of the previous reaction will yield pyruvate and glutamate
  • This cycle is called the Glucose-Alanine cycle (Cahill cycle)
• Glucose-alanine cycle / Cahill cycle

  • Utilization of pyruvate produced by glycolysis in the muscles to carry ammonia to the liver

  A) glucose

  B) 2 pyruvate

  C) 2 alanine

  D) 6

  E) 2

  
• Production of glutamine by most organs

  • Ammonia is also transported in the form of glutamine
  • Glutamate and ammonia are converted to glutamine by glutamine synthetase
  • Investment of 1 ATP
  • Glutamine is carried to the liver where it will be converted back to glutamate
  • In the liver, glutamine is converted into glutamate by the mitochondrial glutaminase
  • The ammonia produced by the reaction can enter the Urea cycle
• Oxidative deamination of glutamate

  • Glutamate dehydrogenase (GDH) catalyzes deamination, not transamination
  • Occurs in the mitochondria
  • This reaction is reversible
  • One NADH will be generated
  • NADPH is used in the reverse reaction
  • The resulting NH4+ enters the urea cycle as one amino group donor

  
• The Urea cycle starts and ends with ornithine; a carrier on which are assembled the carbon and nitrogen atoms that will constitute urea
• Urea cycle (good luck)

  A) glutamate

  B) glutamate dehydrogenase

  C) a-ketoglutarate

  D) NH3

  E) carbamoyl phosphate

  F) citrulline

  G) aspartate

  H) argininosuccinate

  I) fumarate

  J) malate

  K) arginine

  L) urea

  M) ornithine

  
• This part of the urea cycle occurs in the mitochondria

  
• Urea cycle: Mitochondrial phase

  • Reaction catalyzed by CPS1 (carbamoyl phosphate synthetase 1)
  • Condensation of bicarbonate HCO3- with NH3
  • Forms carbamoyl phosphate
  • Requires 2 ATPs

  
• Urea cycle but even worse

  A) ornithine

  B) ornithine transcarbamoylase

  C) citrulline

  D) argininosuccinate synthetase

  E) aspartate

  F) argininosuccinate

  G) argininosuccinase

  H) fumarate

  I) arginine

  J) arginase

  K) urea

  
• The “Krebs bicycle”

  A) Fumarate

  B) aspartate

  
• Urea cycle regulation

  • Nitrogen flux through urea cycle varies based on your diet
• Urea cycle regulation

  Long term regulation through rate of gene synthesis:

  • All four enzymes of the urea cycle, as well as the carbamoyl phosphate synthetase I (CPS1), are synthesized at a higher rate under starving conditions or high- protein diet
  • Under these conditions, proteins are used as a source of fuel and therefore, the production of ammonia is increased
• Urea cycle regulation

  Short term regulation through allosteric control:

  • N-acetylglutamate is an allosteric activator of carbamoyl phosphate synthetase I (CPS1)
  • N-acetylglutamate is synthesized using glutamate and acetyl-CoA
  • This reaction is catalyzed by N-acetylglutamate synthase
  • It requires a high concentration of glutamate
  • **Arginine is an allosteric activator of N-acetylglutamate synthase
• Regulating pathway flux by N-acetylglutamate

  • Arginine acts as an indicator of the level of ammonia in the cell
  • Arginine is synthesized from glutamate

  
• Urea Cycle Summary

  • Urea cycle is confined to the liver
  • Reactions occur in both mitochondria and cytosol
  • Ornithine transport is carried out by the ORC1 translocase
  • Amino groups of urea are derived from NH3 and aspartate, respectively, carbon atom is derived from CO2
  • Carbon skeleton product can be used for gluconeogenesis
• Study when cats don't get arginine from food -> v toxic pee

  • Results in lowering of blood glucose levels
  • Arginine-free diet = catabolism of ingested AAs, especially the glucogenic ones
  • Low concentration of arginine (urea cycle intermediate) slowed the conversion of ammonia to urea
  • Cats synthesize ornithine solely from arginine
  • Arginine deficiency = insufficient amounts of ornithine to feed the urea cycle
  • (a control in the experiment:) Ornithine can be substituted because it is also an intermediate of the urea cycle, since it's a cycle u can form arginine from ornithine
• Most amino acids can be converted to one of seven metabolic intermediates:

  A) a-ketoglutarate

  B) Succinate

  C) fumarate

  D) oxaloacetate

  E) Pyruvate

  F) Acetyl-coA

  G) acetoacetate

  
• Amino acid breakdown

  A) glucogenic

  B) Pyruvate

  C) a-ketoglutarate

  D) Succinyl

  E) oxaloacetate

  F) ketogenic

  G) Acetyl

  H) acetoacetate

  
• Leucine and lysine are ketogenic amino acids
• Private Tim Hall mnemonic trick

  Phenylalanine

  Valine

  Threonine

  Tryptophan

  Isoleucine

  Methionine

  Histidine

  Arginine

  Leucine

  Lysine
• The nitrogen cycle: Nitrogen fixation

  • Inorganic nitrogen (N2) is the most abundant component of earth’s atmosphere
  • Only few microorganisms can reduce N2 to NH3 (NH4+), these are called diazotrophs
  • This reduction process is called Nitrogen fixation
  • This process is catalyzed by the nitrogenase complex
• Nitrogen fixation is a very expensive process: needs 16 ATP
• • Most plant, fungi and bacteria are able to use nitrate ions (NO3-) to generate ammonia (NH3)
  • NO3- is essential for the growth of most plants
• In plants and bacteria, the process by which ammonia will be used to form organic nitrogen-containing molecule like amino acids is called nitrogen assimilation
• Most the nitrogen assimilation is carried out by two

  pathways:

  • Glutamate dehydrogenase (GDH) -> Production of glutamate
  • Glutamine synthetase-glutamine:2-oxoglutarate amido transferase pathway (GS-GOGAT) -> Production of glutamine
• Glutamine synthetase-glutamine:2-oxoglutarate amido transferase pathway (GS-GOGAT)

  A) NH4+

  B) glutamine synthetase

  C) glutamate synthase

  D) a-ketoglutarate

  E) Amino acids

  
• In bacteria and plants, all amino acids can be synthesized from intermediates in glycolysis, the pentose phosphate pathway or the citric acid cycle
• In mammals, non-essential amino acids are synthesized from 4 common intermediates:

  • 3-phosphoglycerate (glycolysis)
  • Pyruvate (glycolysis)
  • α-ketoglutarate (TCA cycle)
  • Oxaloacetate (TCA cycle)
• Non-essential amino acids are synthesized from 4 common intermediates

  A) serine

  B) glycine

  C) cysteine

  D) Alanine

  E) mate

  F) mine

  G) proline

  H) arginine

  I) aspartate

  J) asparagine

  
• A single transamination reaction can convert keto acids into amino acids

  • Alanine transaminase (ALT) -> Glucose alanine cycle
  • Aspartate transaminase (AST)

  
• Amidation reactions using aspartate and glutamate as substrates

  A) asparagine

  B) glutamine