Lectures 7 and 8: Amino acid metabolism

Question 1

What happens to excess amino acids in the diet?

Answer 1

They cannot be stored, so are degraded. The amino group is removed and most is excreted as urea and the carbon skeleton is used as fuel (converted to TCA cycle intermediates).

Question 2

What can amino acids be converted to?

Answer 2

• glucose
• fatty acids (not during starvation)
• ketone bodies (during starvation)

Question 3

How many intermediates are the 20 standard amino acids broken down into? Name them all.

Answer 3

Seven:

Acetoacetyl CoA

Pyruvate

Acetyl CoA

Oxaloacetate

a-ketoglutarate

Succinyl CoA

Fumurate

Question 4

What is significant about the 7 intermediates?

Answer 4

They are all TCA cycle intermediates and can be easily oxidised to produce ATP or used in synthesis.

Question 5

What is phenylketonuria?

Answer 5

A deficiency in the enzyme which hydrolyses phenylalanine to tyrosine (phenylalanine hydroxylase) If it is not treated phenylalanine builds up and can cause brain damage. It can be treated by giving a baby a low protein diet and giving them supplements of the other amino acids for the rest of their life. This disease, along with similar ones for the other amino acids, are screened for in all newborn babies by the NHS.

Question 6

What is a glucogenic amino acid?

Answer 6

Glucogenic amino acids are those that are broken down into oxaloacetate, a-ketoglutarate, succinyl CoA or fumurate. They can by used to synthesise glucose (only relevant in starvation when no glucose is taken up in diet, so muscles must be broken down)

Question 7

What is a ketogenic amino acid?

Answer 7

A ketogenic amino acid cannot be used to synthesis glucose, because it is broken down to give acetyl CoA. Acetyl CoA can be used to make ketone bodies, an alternative fuel to glucose, during starvation.

Question 8

Which amino acids are glucogenic?

Answer 8

Glycine, Alanine, Valine, Cysteine, Serine, Arginine, Proline, Histidine, Methionine, Aspartate, Aspartic Acid, Glutamate and glutamic acid are glucogenic.

Both glucogenic and ketogenic: Phenylalanine, Tyrosine, Tryptophan, Threonine, Isoleucine.

Question 9

Which amino acids are ketogenic?

Answer 9

Lysine and leucine are ketogenic.

Both glucogenic and ketogenic: Phenylalanine, Tyrosine, Tryptophan, Threonine, Isoleucine

Question 10

Which amino acids are broken down into each intermediate?

Answer 10

Acetoacetyl CoA - Leucine, Tyrosine, Phenylalanine Pyruvate - Glycine, Alanine, Tryptophan, Serine, Cysteine Acetyl CoA - Acetoacetyl CoA and pyruvate lead to this a-ketoglutarate - Arginine, Proline, Histidine Succinyl CoA - Methionine, Isoleucine, Valine Fumurate - Tyrosine Oxaloacetate - Aspartate, Asparagine

Question 11

By which process is the alpha amino group removed from the amino acid?

Answer 11

Transamination, transferring the amino group to an a-keto acid

Question 12

Describe how the amino group is removed from amino acids.

Answer 12

a-amino acid 1 + a-keto acid 1 —> a-keto acid 2 + a-amino acid 2 The amino group has been transferred from the original amino acid to an a-keto acid, converting the amino acid to an a-keto acid and building up the original a-keto acid into an amino acid by adding the NH3+. For example: a-amino acid 1 + a-keto glutarate —> a-keto acid 2 + glutamate

Question 13

What is formed when aspartate is deaminated?

Answer 13

oxaloacetate

Question 14

What is formed when alanine is deaminated?

Answer 14

Pyruvate

Question 15

Which enxymes catalyse transamination reactions?

Answer 15

aminotransferases, e.g. alanine aminotransferase, aspartate aminotransferase

Question 16

What do aminotransferases use as a cofactor?

Answer 16

Pyridoxal phosphate (derived from pyridoxine, aka vitamin B6), which is a carrier of amino groups. It is also used by glycogen phosphorylase, which is much less common a usage.

Question 17

What are the consequences of the reversibility of the transamination reactions?

Answer 17

The transamination reactions are fully reversible reactions, so they operate at equilibrium and the direction of the reaction depends on the concentrations of reactants in the cell.

e.g. aspartate high - degraded to oxaloacetate

aspartate low - synthesised from oxaloacetate

There is no control by enzymes, it is all done by mass action (concentrations of reactants)

Question 18

Describe the mechanism of the amino transferase mechamism.

Answer 18

The reactive aldehyde group on the cofactor pyridoxal phosphate (abbreviated to PLP) reacts with the amino NH2 group in the amino acid 1 and produces a Schiff base intermediate, with the loss of water.

Then internal rearrangement of electrons occurs in the Schiff base-R1, alternating between the quinonoid intermediate and the carbanion-R1.

Hydrolysis of the Schiff base leaves an a-keto acid-R1 and PLP with the amino group attached (pyridoxamine phosphate) .

Then the pyridoxamine phosphate is converted back to PLP by another a-keto acid, which is itself converted to an amino acid.

Question 19

What is the significance of glutamate in amino acid degradation?

Answer 19

All the amino groups which are transferred from amino acids being degraded are eventually added to a-ketoglutarate to from glutamate. Glutamate then undergoes an oxidative deamination, releasing free ammonia. In the process, glutamate is converted to a-keto glutarate via a Schiff base intermediate. The mammalian enzyme requires a cofactor, which can either be NAD+ or NADP+, which aids the oxidation to the Schiff base, which then undergoes hydrolysis, losing ammonia in the step to a-keto glutarate.

Question 20

The glutamate dehydrogenase reaction is completely reversible. What drives it forward?

Answer 20

The production of urea from ammonia. Removing ammonia from the reaction drives the reaction towards the products.

Question 21

Where does the Urea Cycle take place?

Answer 21

In the liver

Question 22

Which cellular compartment(s) does the Urea Cycle take place in?

Answer 22

The mitochondrial matrix and the cytosol

Question 23

Give the steps of the Urea Cycle.

Answer 23

1) Free ammonia and bicarbonate enter the mitochondrial matrix and react with 2 ATP to form Carbamoyl phosphate. The reaction is catalysed by carbamoyl phosphate synthase and releases 2 ADP, Pi and 3 H+.
2) Carbamoyl phosphate transfers the carbonyl group onto ornithine, which is an amino acid not found in proteins. This releases Pi and produces L-Citruiline.
3) L-Citruiline leaves the mitochondrial matrix into the cytosol, where a condensation reaction occurs between the carbonyl group of L-Citruiline and the amino group of L-aspartate. This is where the second NH2 in urea comes from. This uses the equivalent of 2 ATP (actually ATP –> AMP + PPi) and produces Argininosuccinate. This reaction is catalysed by Argininosuccinate synthase.
4) There is cleavage releasing fumurate and leaving L- arginine to continue in the cycle. This reaction is phosphorylated by Arginiinosuccinate lyase.
5) L-Arginine is cleaved, releasing urea. Ornithine is regenerated and enters the mitochondria again to accept another carbamoyl to begin another cycle. This reaction is catalysed by Arginase.

Question 24

What is the chemical structure of urea?

Answer 24

Question 25

Where do the two amino groups in urea come from?

Answer 25

The first NH2 comes from free ammonia produced when glutamate is converted to a-keto glutarate via a Schiff’s base intermediate in an oxidation then hydrolysis reaction.

The second NH2 comes from L-aspartate, which enters the urea cycle in the 3rd reaction when its NH2 group reacts with L-Citruiline’s carbonyl group.

Question 26

What is the enzyme for the first reaction of urea synthesis in the Urea Cycle?

Answer 26

Carbamoyl phosphate synthase 1

Question 27

Describe the steps of the first reaction in synthesising urea in the Urea Cycle.

Answer 27

1) Bicarbonate is phosphorylated by ATP, giving carbonyl phosphate
2) The phosphoryl group on carbonyl phosphate is displaced by ammonia, which attacks the carbonyl group. This gives carbamate.
3) Carbamate is phosphorylated using a second ATP, giving Carbamoyl phosphate.

Carbamoyl acts as an activated carbonyl donor in the Urea Cycle.

Question 28

By which intermediate is the Urea Cycle linked to the TCA cycle?

Answer 28

Fumurate

Question 29

Give the balanced reaction of Urea Cycle.

Answer 29

Bicarbonate (HCO3-) + NH4+ + 3 ATP + Aspartate + H2O

—>

Urea + 2 ADP + 2 Pi + AMP + PPi + Fumurate

Question 30

How is the 4 ATP cost of removing nitrogen from amino acids justified?

Answer 30

Nitrogen is toxic to the cell so must be removed. Also, one molecule of fumurate is formed, which can enter the TCA cycle and make up for 2.5 moles of the ATP used.

Question 31

What is the role of glutamine in nitrogen metabolism?

Answer 31

Not all NH4+ should be excreted as urea, because some is needed for synthesis, e.g. amino acids, nucleotides, amino sugars (for glycolipids/proteins).

Therefore, some nitrogen is encorporated into glutamine, which acts as a non-toxic carrier of nitrogen, so is used to carry it between tissues and travels via bloodstream.

Glutamate is the main carrier in cells, but can’t pass the membranes easily, so it is converted to glutamine, which can because it is non-polar.

Glutamine is also used as a nitrogen donor in many synthetic reactions. It has a higher concentration in the cells than other amino acids, because of its special role.

Question 32

How is glutamine converted to glutamate?

Answer 32

Glutamate + NH4+ + ATP —> Glutamine + ADP + Pi

This reaction is catalysed by glutamine synthase.

Question 33

What do the precursors of amino acids all have in common?

Answer 33

There are intermediates in glycolysis or the TCA cycle

Question 34

Which amino acids are absoutley essential?

Answer 34

Histidine, Phenylalanine, Tryptophan, Valine, Leucine, Isoleucine

Question 35

Which amino acids are not synthesis at a high enough rate to form sufficeint quantities so must also be in the diet?

Answer 35

Methionine and Lydine

Question 36

How are the essential amino acids obtained?

Answer 36

In the diet from plants and bacteria

Question 37

What can a) aspartic acid, b) alanine and c) glutamic acid be synthesised from via transamination reactions?

Answer 37

a) oxaloacetate
b) pyruvate
c) a-ketoglutarate

Question 38

Why are alanine, aspartic acid and glutamic acid the only amino acids useful in synthesis of TCA cycle intermediates?

Answer 38

Because keto acids are produced in other reactions, therefore are at high concentrations in the cell ???

Question 39

Give three important roles of glutamate.

Answer 39

1) In aminotransferase reactions, an a amino group can be transferred onto a-ketoglutarate to form glutamate
2) Glutamate dehydrogenase releases free ammonia for urea synthesis or glutamine synthesis.
3) Glutamine synthase forms glutamine from glutamic acid and free ammonia for non-toxic transport of nitrogen between tissus

Question 40

What are ketone bodies synthesised from?

Answer 40

Acetyl CoA

Question 41

Explain why ketone bodies are produced when carbohydrate levels are low?

Answer 41

When carbohydrate levels are low, gluconeogenesis occurs, using up oxaloacetate. This means that the high levels of acetyl CoA released from fatty acid degradation cannot react with oxaloacetate to form citrate to enter the TCA cycle. Therefore the remaining acetyl CoA must enter another pathway so that it is not wasted. This pathway leads to the formation of ketone bodies, an alternative fuel to glucose which can be used in the brain.

Question 42

Where are ketone bodies synthesised?

Answer 42

In the mitochondrial matrix in liver cells

Question 43

Which tissue is particularly good at using ketone bodies?

Answer 43

Heart muscle

Question 44

When is the synthesis of ketone bodies greatly increased?

Answer 44

During starvation

Question 45

Why can’t the brain break down fatty acids to produce glucose?

Answer 45

Fatty acids cannot pass the blood-brain barrier

Question 46

Which disease is associated with high levels of ketone bodies in non-starvation conditions?

Answer 46

Untreated diabetes.

High blood glucose but no insulin, so nothing is done with the excess glucose, so ketone bodies are used instead.

Production of acetone (a ketone body) is a diagnostic test of diabetes.

Question 47

Describe the steps of ketone body synthesis.

Answer 47

1) Acetyl CoA loses CoA-SH in a reaction catalysed by beta-Ketothiolase, forming Acetoacetyl-CoA
2) Acetoacetyl-CoA is converted to beta-Hydrocy-Beta-methylglutaryl-CoA (HMG-CoA), by HMG-CoA synthase, with acetyl CoA being simultaneously converted to CoA-SH.
3) Then HMG-CoA loses acetyl CoA in a lysis reaction catalysed by HMG-CoA lyase to produce Acetoacetate, a ketone body.
4) Acetoacetate can produce other ketone bodies, such as Acetone, by losing CO2, or beta-Hydroxybutyrate, with NADH +H+ simultaneously being converted to NAD+ (this reaction is catalysed by beta-hydroxylbutyrate dehydrogenase).

Question 48

You’re doing really well :) Congrats on your progress :)

Answer 48

Question 49

Describe the steps of ketone body degradation.

Answer 49

1) D-beta-Hydroxybutyrate is converted to Acetoacetate by betoa-hydroxybutyrate dehydrogenase, with the simultaneous conversion of NAD+ to NADH + H+.
2) Acetoacetate is converted to Acetoacetyl-CoA by 3-ketoacyl-CoA transferase, with the simultaneous conversion of Succinyl-CoA to succinate.
3) Acetoacetyl-CoA is converted to 2 molecules of acetyl CoA by thiolase, with H-SCoA being added during the reaction.

Question 50

What is HMG-CoA important in?

Answer 50

Ketone body synthesis and cholesterol synthesis

Question 51

Why can animals not synthesise glucose from acetyl CoA?

Answer 51

The 2 carbons in acetyl CoA are released as carbon dioxide, so there is no net synthesis of oxaloacetate or glucose from acetyl CoA.

Question 52

How can plants and bacteria make glucose from acetyl CoA?

Answer 52

Plants and bacteria have the glyoxalate cycle, which requires two extra enzymes not found in animals: isocitrate lyase and malate synthase. The pathway bypass the 2 steps where CO2 is released.

Isocitrate lyase takes one molecule of isocitrate and splits in into succinate and glyoxaloacetate.

Malate synthase combines glyoxalate and acetyl CoA to form malate.

Question 53

Why is cholesterol important?

Answer 53

Cholesterol is an important component of cell membranes in animals, improving stability.

Cholesterol is also important in the synthesis of steriod hormones.

Question 54

How is HMG-CoA important in cholesterol synthesis?

Answer 54

HMG-CoA is converted to Mevalonate by the enzyme HMG-CoA reductase, with the simultaneous conversion of 2 NADPH + 2 H+ to 2 NADP+ and the release of CoA-SH. This is the commited step of cholesterol synthesis.

Mevalonate is important in cholesterol synthesis because it is converted to an activated isoprene (isoprene with 2 phosphate groups attached), which is converted to squalene and then cholesterol.

Question 55

Describe how a widely taken drug can prevent heart disease by reducing cholesterol in the blood.

Answer 55

Statin drugs inhibit cholesterol synthesis by inhibiting HMG-CoA reductase, which catalyses the committed step of cholesterol synthesis.