Part 1. Amino Acid Degradation and Urea Cycle

Question 1

What is the amino acid pool?

Inputs and utilization

Answer 1

Protein turnover (defective damaged, not needed proteins), dietary proteins, and amino acid biosynthesis are the input

The utilization of amino acid pool is synthesis of proteins, synthesis of nitrogenous molecules (purines, pyrimidines), and degradation of amino acids carbon skeleton

If a molecule can be converted to acetyl CoA, then ketone bodies, fatty acids, and steroids can be made
Slide 3 mar 8

Question 2

How are dietary proteins digested?

Answer 2

Proteins are too large generally to be absorbed intact so are digested into amino acids and di and tri peptides

Proteolysis- hydrolysis cleavage of proteins by proteases in stomach and intestine

Slide 5 Mar 8

Question 3

What are the steps of protein digestion?

Answer 3

1. The mouth sets the path for the beginning of digestion, chewing causes neural effects that stimulate gastric juice production and release of peptide hormone production
2. True digestion begins in the stomach (more protein added, more gastric juice pH 1.5-3)
   Pepsin used to break up proteins
3. Small intestine is where protein digestion is completed, bicarbonate is major component as base buffer between the gastric juice and other compounds
   Proteases act upon the broken down molecules to digest them (need denatured proteins)
4. Then amino acids absorbed to epithelial cells and moved to blood by portal vein

Question 4

What is the gastroesophageal reflux disease (GERD)?
Heartburn!
Medications?

Answer 4

The K+/H+ pump in the membrane of specialized stomach cells pumps protons into the stomach in exchange for K+ at the expense of ATP hydrolysis, this generates acidic environment and releases heat
cAMP levels rise then PKA (protein kinase A) phosphorylates the proton pump and moved them to surface of cell
If the pump is overactive or the esophageal sphincter is weak, GERD is the result (add tums if over production of acid, histamine blocker blocks one half of pathway, and direct proton pump inhibitor)

Slides 8-9 Mar 8

Question 5

What are zymogens?

Answer 5

All proteolytic enzymes are secreted as inactive zymogens
They are activated by proteolysis which allows for proper folding of the enzyme

Proteolytic enzymes are stored as zymogens so that they don’t breakdown proteins in the cells where they are made and stored

Slide 11 Mar 8 pepsinogen converted to active form (red catalytically inactive)
Slide 12 Mar 8

Question 6

Why are there so many different proteolytic enzymes?

What are pepsin and trypsin examples?

Answer 6

They have different substrate specificities to allow proteins to be cut into much smaller fragments rapidly
Substrates have substrate specificity and side chains can be very different in amino acids

Pepsin- preferentially cleaves peptide bonds between hydrophobic amino acids and aromatic amino acids
Trypsin- cleaves peptide bonds following Arg or Lys residue

Slide 14 Mar 8

Question 7

What is the 3 steps of the fate of the amino group of amino acids?

Answer 7

1. Transanimation
   α-amino acids and α-ketoglutarate are converted to α-keto acids and L-glutamate
2. Oxidative deamination
   L-glutamate is converted to α-ketoglutarate and produces NH4+ (ammonia)
3. Urea cycle
   NH4+ and CO2, Asp are converted to urea in the liver then moves to blood then kidney then urine

Transdeamination reaction= transamination + oxidative deamination reactions

Slide 5 Mar 11

Question 8

What are the steps in transamination?

Answer 8

Enzyme- aminotransferase
Coenzyme- pyridoxal phosphate (PLP)
L amino acid transfers amino group to α-ketoglutarate (L-glutamate)

Slide 6-7 Mar 11

Question 9

What are the two specific aminotransferases and their amino acid/Keto-acid pairings?

Answer 9

Alanine transaminase- glutamate + pyruvate = alanine + α-ketoglutarate

AST - glutamate + oxaloacetate = asparate + α-ketoglutarate

Slide 8 Mar 11

3rd one is aminotransferase with its four

Question 10

What is the fate of glutamate in the liver?

Answer 10

Transported to mitochondria and undergoes oxidative deamination to release ammonia
Amino acids -> glutamate by aminotransferase
Glutamate+α-keto acid -> glutamate (in the mitochondrion)
Glutamate -> α-ketoglutarate + NH4+

Slide 9 Mar 11

Question 11

What are the steps and enzyme uses in the oxidative deamination of glutamate?
Activators and inhibitors?

Answer 11

Enzyme- L-glutamate dehydrogenase
Coenzyme- NAD+ or NADP+
ADP activates and GTP inhibits

Removes amino group from glutamate as NH4+
High concentration of NH4+ is toxic
NH4+ is converted to urea in the liver

Slide 10 Mar 11

Question 12

What are the 2 ways that ammonia produced in non liver tissues get to the liver so it can be converted to urea?

Answer 12

1. In most tissues: NH4+ reacts with glutamate to form glutamine (by enzyme glutamine synthetase)
   Glutamine is a non toxic storage and transport for NH4+ by the blood
   NH4+ is released by glutamine by cleavage by the enzyme glutaminase
2. In muscle: glucose alanine cycle, glutamate is formed by transamination reactions, glutamate then transfers it’s amino group to pyruvate to form alanine which can leave muscles to the blood to liver where transamination occurs again to release the amino group as ammonium

Slides 11-12 Mar 11

Question 13

What is the overview and goals of amino group catabolism in the liver?

Answer 13

Slide 2 Mar 13
Goal is to keep levels of free NH4+ low (high levels are harmful to CNS), and to get NH4+ to the liver so it can be used to produce urea which is non toxic and excretable

NH4+ is transported to liver as alanine and go

Question 14

What is the urea cycle?

Answer 14

Cyclical pathway whereby excess NH4+ is converted to urea
(Memorize NH4+ structure slide 3 mar 13)
Occurs in liver through a series of reactions in the cytosol and mitochondria (cytosol is enzymes are present in a cluster)

Urea is secreted into the blood, filtered by kidneys, then excreted in urine

Costs 3 ATP

Examples slides 4-6 Mar 13
Overview slide 7 Mar 13

Question 15

What is carbamoyl phosphate synthesis?

It’s enzyme?

Answer 15

Ammonia + bicarbonate -> carbamoyl phosphate
Enzyme: carbamoyl phosphate synthetase I
2 ATP in and 2 ADP + Pi out

THIS IS RATE LIMITING STEP IN UREA BIOSYNTHESIS

Requires N-acetylglutamate as allosteric activator

Slides 8-9 Mar 13

Question 16

Why is carbamoyl phosphate synthesis I allostericallt regulated by N-acetylglutamate?

Answer 16

N-acetylglutamate is synthesized from glutamate and acetyl CoA by N-acetylglutamate synthase, a reaction stimulated by arginine

Amino acids provide most of the excess ammonium, thus after a meal containing protein, ammonium production also increases

Therefore at the same time that ammonium levels are going up, the production of N-acetylglutamate goes up which activates CPS-I (first and rate limiting step in urea synthesis)

Question 17

What are the 4 enzymes in the second part of the urea cycle?

Answer 17

Reaction 1: Orn transcarbamoylase
Reaction 2: argininosuccinate synthetase
Reaction 3: agrininosuccinate lyase
Reaction 4: arginase
Enzymes 2-4 are clustered (rapid reaction rates, intermediates remain in complex, concentration of intermediates is high, and intermediates do not undergo side reactions)

Slide 12 Mar 13
Know all enzymes and intermediates here

Question 18

What is the immediate source of urea?

Answer 18

Arginine

Which is produced by arginase

Question 19

What is Kreb’s Bicycle?

Answer 19

Works along side the urea cycle with argininosuccinate

Urea cycle and TCA cycle are integrated by aspartate and fumarate (Kreb’s bicycle)

Slide 13 Mar 13

Question 20

What is the overall reaction of Urea synthesis?

Answer 20

Asp + NH4 + HCO3 + 3ATP -> urea + fumarate + 2ADP + AMP + 2Pi + PPi

Question 21

How do ammoniotelic organisms dispose of excess nitrogen?

How do uricotelic organisms dispose of nitrogen?

Answer 21

Release ammonia directly; usually aquatic organisms

Uricotelic- uric acid, a purine metabolite, is only slightly soluble in water and this requires very little water to get ride of it
Generally used by organisms that can’t afford to lose water (reptiles and birds)

Question 22

What are the 4 sources of free NH4+?

Answer 22

1. Major source is dietary and body protein- transdeamination of amino acids in liver, serine and threonine dehydratase in liver, glutaminase in liver, amino groups from other tissues all release NH4+
2. Kidney- Gln -> NH4+ + Glu
   Gln -> NH4+ + α-KG
3. Amines - monoamines, neurotransmitters
4. Purine and pyrimidine catabolism

Slides 17-20 Mar 13

Question 23

What are the 3 mechanisms that keep ammonium levels in blood low?

Answer 23

1. Formation of urea by urea cycle
2. Formation of glutamate in muscle by glutamate dehydrogenase
3. Formation of glutamine in most tissues by glutamine synthetase

Question 24

What is hyperammonemia?

Symptoms?

Answer 24

Ammonia is toxic to CNS
Brain tries to reduce levels by synthesizing glutamine from glutamate; this leads to increased levels of glutamine, but depletion of glutamate. Both are problematic
Symptoms: all symptoms attribute to CNS in a negative way

Causes: can result from liver damage (aquired hyperammonia) or genetic factors (congenital form)
Goes up after eating a high protein diet

Question 25

What is glutamine and glutamate?

Answer 25

Glutamine is an osmolarity in astrocytes, which causes an uptake of water and this brain swelling
Glutamate is itself a neurotransmitter, but also a substrate for the synthesis of a second neurotransmitter, GABA, so neurotransmitter function is affected

Question 26

What is acquired hyperammonemia?

What is congenital hyperammonemia?

Answer 26

Acquired- liver damage can result from viral hepatitis, hepatoxins such as alcohol
When liver becomes cirrhotic, collateral circulation develops which bypasses the liver, leading to elevated ammonia
Congenital- symptoms appear shortly after birth
Defects in the genes coding for all five of the urea cycle enzymes have been described that lead to hyperammonemia
Defects in arginase is least severe cause if 3 amino groups and can be filtered out of kidneys

Question 27

What target glutamine?

Answer 27

Not essential amino acid
Main carrier of ammonium from peripheral tissues
Contains 2 amino groups