Lecture 35: Amino Acid Catabolism/Urea Cycle

What are the 2 ways proteins are degraded?

• ATP-dependent process with ubiquitin

• ATP-independent process in lysosomes

When are amino acids oxidized/catabolized in animals?

• during normal protein turnover

• after protein-rich meal

• during prolonged fasting, starvation, and uncontrolled diabetes (mostly from degradation of muscle protein)

What lowers pH (1-2.5) in the stomach?

HCl secretion

What results from low pH?

• denaturation of proteins

• hydrolysis of peptide bonds by pepsin

• kills microorganism

What raises the pH in the intestine?

secretion of bicarbonate by the pancreas

What are the pancreatic proteases?

• trypsin

• chymotrypsin

• carboxypeptidases

What are the intestinal proteases?

aminopeptidases

What happens after amino acids are absorbed by intestinal epithelia?

amino acids are released into the bloodstream via the hepatic portal vein

What is the process for dietary protein digestion?

1. HCl denatures proteins

2. pepsin begins breaking down proteins into shorter chains

3. pancreatic enzymes break down proteins further

4. peptidases from the intestinal lining further break down peptides into dipeptides and free amino acids

5. amino acids are absorbed by intestinal epithelia

6. amino acids are released into the bloodstream via the hepatic portal vein

What is the first pass effect?

substances absorbed in the GI system perfuse the liver before continuing to the rest of the circulation

What is the primary site for amino acid catabolism?

the liver

What is important to know about the liver?

• double blood supply:

  • heart

  • hepatic portal vein

How do cells take up amino acids?

by active transport

What does active transport of amino acids lead to?

a higher amino acid concentration in the cytoplasm than outside the cell

Describe cystinuria.

• a disorder of the proximal tubule’s reabsorption of filtered cystine and dibasic amino acids (lysine, ornithine, and arginine)

• inability to reabsorb cystine leads to accumulation and subsequent precipitation of stones of cystine in the urinary tract

• defect in a specific amino acid transporter

When amino acids are catabolized, what is the first step?

deamination

True or False: There are different fates for amino groups and carbon skeletons after amino acid degradation.

true

What are amino groups after degradation used for?

• biosynthetic reactions

• excreted

True or False: The method of excretion of nitrogen from excess animo groups varies by organism.

true

Aquatic animals can excrete NH₄⁺ directly, but this is too toxic for terrestrial organisms to accumulates. How do terrestrial organisms typically excrete nitrogen?

as urea or uric acid

True or False: Urea and uric acid have highly oxidized carbons—meaning there’s not a lot of energy left.

true

In vertebrates, where does most amino acid degradation occur?

the liver

Where does transamination occur?

the cytosol

Amino groups are moved to an acceptor molecule—converting the starting amino acid to what?

a-keto acid

What will alanine become?

pyruvate

What will glutamate become?

a-ketoglutarate

For many amino acids, what is the amino group acceptor for transamination?

a-ketoglutarate

What does amino-transferase do?

• converts a-ketoglutarate to L-glutamate 

• converts L-amino acid to a-keto acid

What cofactor does amino-transferase need?

PLP

True or False: Glutamate can act as the amino group donor for biosynthetic reactions or for nitrogen excretion.

true

Is transamination irreversible or reversible?

reversible

True or False: Glutamate and glutamine have central roles in the metabolism of the amino groups of amino acids; these amino acids are present at higher concentrations than most other amino acids.

true

Most tissues transfer excess amino groups from glutamine to what?

to blood to the liver cells

In liver cells, what does glutaminase do?

deaminates glutamine to glutamate and ammonia (NH₄⁺)

Muscles also transfer excess amino groups from alanine to where?

to blood to the liver cells

In liver cells, amino groups are transferred from alanine to what?

glutamate

In muscles, what does alanine aminotransferase do?

converts glutamate to a-ketoglutarate

In the liver, what does alanine aminotransferase do?

converts a-ketoglutarate to glutamate 

True or False: Muscles (and some other tissues) catabolize amino acids for fuel.

true

What is ammonia in muscle cells transferred to and what does this make?

transferred to pyruvate to make alanine, which is sent via blood to the liver

What does the liver do (regarding the glucose-alanine cycle)?

liver deaminates alanine and uses pyruvate to re-synthesize glucose via gluconeogenesis

What is the pyridoxal phosphate cofactor derived from?

vitamin B₆/pyridoxine 

True or False: Transaminases/aminotransferases require PLP as a cofactor, but the cofactor is typically in the pyridoxamine phosphate form. PLP is a versatile cofactor.

true

How are amino acids linked to PLP?

via the Schiff base

True or False: PLP has an electron sink just like NAD⁺ does—making the a,b, and c cleavages possible.

true

What is the transaminase mechanism?

1. the PLP cofactor forms a Schiff base (aldimine) with the E-amino group of a lysine from the enzyme

2. PLP-enzyme Schiff base exchanges with incoming amino acid -NH

What are the clinical tests for transaminases?

• AST: aspartate aminotransferase (serum glutamate-OAA transaminase)

• ALT: alanine aminotransferase (serum glutamate-pyruvate transaminase)

True or False: In the liver, glutamate is also converted to a-ketoglutarate via oxidative deamination—releasing the amino groups as ammonia. Ammonia is converted ultimately to urea via the urea cycle.

true

What enzyme is needed for oxidative deamination?

glutamate dehydrogenase

What does oxidative deamination of glutamate yield?

• a-ketoglutarate 

• ammonia

• NADH

Where does oxidative deamination occur?

in the mitochondrial matrix in liver cells

True or False: In hepatocyte cytosol, most excess amino groups are transferred to a-ketoglutarate.

true

Where is urea produced?

in the liver

Ammonia is converted into urea via how many enzymatic steps?

5 steps (4 of which comprise the urea cycle)

True or False: Portions of the urea cycle occur in the cytosol and in the mitochondrial matrix.

true

What does the nitrogen in urea come from?

1 from ammonia and one from Asp

True or False: Ornithine is a true catalyst, in contrast to OAA in the TCA cycle.

true

What occurs prior to the urea cycle?

synthesis of carbamoyl phosphate in the mitochondrial matrix

How many active sites does carbamoyl phosphate synthetase I have? How are they connected?

• 3 active sites

• connected through “substrate channeling”

What is substrate channeling?

the product from one active site is channeled to the other active site and so forth

Where does carbamoyl phosphate synthetase I act?

the mitochondrial matrix

What is the carbamoyl phosphate synthetase I mechanism?

1. ATP phosphorylates bicarbonate

2. ammonia displaces phosphoryl group—generating carbamate

3. carbamate is phosphorylated—yielding carbamoyl phosphate

True or False: In the carbamoyl phosphate synthetase I reaction, two activation steps are used by this enzyme to generate carbamoyl phosphate. 

true

True or False: Carbamoyl phosphate synthetase I doesn’t employ biotin even though it uses bicarbonate.

true

Describe step 1 of the urea cycle.

• substrate(s): carbamoyl phosphate and ornithine

• coactivator(s): none

• enzyme: ornithine transcarbamoylase

• product(s): citrulline and P_i

Describe the mechanism for ornithine transcarbamoylase.

the amino group on ornithine makes a nucleophilic attack on the carbonyl of carbamoyl phosphate

After citrulline is produced, where is it transported?

the cytosol

Describe step 2 of the urea cycle.

• substrate(s): citrulline

• coactivator(s): ATP

• enzyme: argininosuccinate synthetase

• product(s): AMP, PP_i, and argininosuccinate

Describe the mechanism for argininosuccinate synthetase.

1. rearrangement leads to addition of AMP—activating the carbonyl oxygen of citrulline

2. aspartate addition is facilitated by displacement of AMP

Describe step 3 of the urea cycle.

• substrate(s): arginiosuccinate

• coactivator(s): none

• enzyme: argininosuccinase

• product(s): arginine and fumarate

Where does ornithine transcarbamoylase take place?

the cytosol

Where does argininosuccinase take place?

the cytosol

Describe the mechanism for argininosuccinase.

a proton is abstracted and the carbon-nitrogen bond is cleaved

True or False: After the argininosuccinase reaction, arginine carries both amino groups (one originally from NH₄⁺, one from aspartate).

true

Describe step 4 of the urea cycle.

• substrate(s): arginine

• coactivator(s): H₂O

• enzyme: arginase

• product(s): ornithine and urea

Where does the arginase step take place?

the cytosol

Where do the various atoms come from in urea?

• 1 N: comes from aspartate

• O: comes from water

• 1 N: comes from ammonia

• C: comes from bicarbonate

What is the starting point of the urea cycle?

ornithine

Which cycle is the urea cycle linked to?

the TCA cycle

How is the urea cycle linked to the TCA cycle?

through cytosolic isozymes and transport systems

What is the short-term regulation of the urea cycle?

N-acetylglutamate allosterically regulates carboamoyl phosphate synthetase I

What is the long-term regulation of the urea cycle?

increased urea enzyme synthesis

What can compromised liver function or genetic deficiencies in the urea cycle enzymes (or in the product of N-acetylglutamate) lead to?

hyperammonemia

What is ammonia especially toxic to?

the CNS

What does acute ammonia toxicity cause?

• lethargy

• vomiting

• cerebral edema

• coma

• death

What does chronic ammonia toxicity cause?

• developmental delay

• mental retardation

• progressive liver damage

What are the normal serum ammonia amounts?

10 to 14 micro molars

What is the serum ammonia amount in hyperammonemia?

the levels can exceed 1000 micro molars

What is the mechanism of ammonia toxicity?

1. glutamate dehydrogenase reaction is shifted toward synthesis of glutamate—resulting in the depletion of the citric acid cycle intermediates and deprivation of brain energy

2. glutamine is the main source of glutamate and is produced by the glial cells

3. ammonia inhibits the activity of glutaminase—resulting in neurotransmitter deficiency

What are the treatments for urea cycle disorders?

• immediate hemodialysis to remove ammonia from the blood

• low-protein diet

• administration of substances to promote excretion of excess amino groups in stoichiometric amounts

Describe argininosuccinase deficiency.

• administer stoichiometric amounts of arginine

  • argininosuccinate is eliminated in the urine

Describe ornithine trascarbamoylase deficiency.

• X-linked disorder

• administer benzoate and phenylbutyrate

• hippurate and phenylacetylglutamine is eliminated in the urine

What is the stoichiometry of the urea cycle?

aspartate + NH₃ + CO₂ → urea + fumarate

What is the energy cost of the urea cycle?

4 ATPs

Where does the urea cycle take place?

cytoplasm and mitochondrial matrix of liver cells

Which amino acids are exclusively ketogenic?

• Leu

• Lys

Which amino acids are exlusively glucogenic?

• alanine

• cysteine

• glycine

• serine

• asparagine

• aspartate

• glutamate

• histidine

• proline

• methionine

• valine

Which amino acids are both ketogenic and glucogenic?

• phenylalanine

• tryptophan

• tyrosine

• isoleucine

• threonine