Amino Acid Catabolism

Question 1

What happens to the NH3 group in amino acids?

What do these tissues use amino acids for?

Skeletal muscle
Liver
Tissues
Small intestine
Brain

Answer 1

Group is degraded by enzymes & taken to liver into urea to turn intoxic

1. Uses branched amino acids as fuel- Ile, Leu, Val
2. uses as fuel & makes urea
3. use AA as fuel- Ala, Glu, Gln & send their N waste to liver
4. use AA for digestion
5. doesn’t use AA- only glucose & ketone bodies

Question 2

What does an aminotransferase do?

What products does alanine always produce on its transamination? What is significant about the other product? What cycle is it also involved in?

What happens in the transamination of alanine with alanine transaminase & alphaketoglutamate?

What is great about the reaction of transamination?

Answer 2

Enzyme that transfers amino group from alpha amino acid to an alpha keto acid to produce a new alpha amino acid & other product
AA & ketoacid -> ketoacid & AA

alpha amino acid & pyruvate (ketoacid)- pyruvate is a product of glycolysis- made into acetyl coA in citric acid cycle

forms glutamate & pyruvate

reversible as G=0 and k=1

Question 3

What product arises from glutamate transamination? What enzyme is used?

What happens to leucine in transamination?

Why is leucine different to glutamate & alanine in transamination?

how is the alpha ketoacid esterified to acetyl coA? what changes with this reaction?

Answer 3

alpha keto glutamate = intermediate in cyclic acid cycle. glutamate transferase/dehydrogenase

Leucine -> ketoacid -> acetylcoA (esterified)

leucine is a ketogenic amino acid (makes ketone bodies) as can’t be made into glucose as forms acetyl-coA
pyruvate & alpha-ketoglutamate can be used to make glucose as alanine & glutamate are glucagenic AAs

oxidised (NADH/FADH2), hydrated & cleaved: irreversible- stays in citric acid cycle

Question 4

What happens to amino acids in the FED state generally?

How do the following tissues catabolise amino acids in the FED state?

muscle
brain
liver
small intestine

How do the following tissues catabolise amino acids in the STARVING state?

muscle
brain
liver
small intestine

why is a lot of AA catabolism inhibited when starving?

what are amino acids mainly converted to?

which organ is the best for using amino acids?

Answer 4

excess amino acids can’t be stored- so are either used as fuel or alpha keto acids converted into triglyceroles or glucose

1. takes up branched amino acids as fuel- ile, leu (coA), val
2. doesn’t metabolise AA uses glucose as a fuel in fed state
3. uses AA as fuel & uses remaining carbon structures to form glucose or fat
4. uses excess amino acids as fuel for digestion & circulation- mainly gutamine
5. muscle breaks down glucose -> pyruvate -> glucose
6. uses glucose & ketone bodies- replaced by using excess carbon structures from AA
7. uses mainly ketogenic AA (leucine) & converts into glucose & ketone bodies
8. uses AA from blood circulation especially glutamine & nitrogen waste sent liver

there’s no storage for proteins

same products as glycolysis, fatty acid oxidation & citric acid cycle intermediate

liver- in fed state avoids losing energy & in starving makes glucose

Question 5

What is the difference between transamination and deamination?

What cofactor do transaminases have & where are they from?

What do the enzymes broadly do?

What is the equation for oxidative transamination of glutamate? What enzyme is used?

What is the equation for oxidative deamination of glutamate? What enzyme is used? What is the co-enzyme?

What happens to the nitrogen by-product?

What does both transamination & deamination of glutamate allow?

Answer 5

Transamination = transfer of amino group to another (e.g keto/oxo acid)
Deamination = removal of an amino group

Pyridoxal phosphate- B6

Interconvert amino acids & funnel them either into aspartate (in urea cycle) or glutamate (undergoes oxidative phosphorylation)

Pyruvate + glutamate -> alanine + 2-oxoglutarate
aminotransaminase

glutamate + NAD+ + H2O -> 2-oxoglutarate & NH4+ + NADH + H+
glutamate dehydrogenase
NAD+ or NADP+

NH4+ transported to liver as glutamine & converted into carbomyl phosphate to enter the urea cycle

Removal of its amino groups means the carbon skeleton can be oxidised

Question 6

How is the heart extract prepared in 3 steps?

What is removed in the dialysis step? How?

How are the amino acids & oxo acids separated?

How are they visualised?

Answer 6

Minced
Homogenised 20,000g x 20 minutes in ice cold buffer
Supernatant is dialysed exhaustively (changes to dialysis buffer) in ice cold buffer 0.05mol L-1 phosphate, pH 7.4

amino acids
NAD+
oxo acids
We place the liquid inside a bag made of a membrane with very small pores. Only the smallest molecules eg amino acids, oxo acids and NAD would be able to migrate through the pores. All other soluble components would be retained.

TLC on 2 different plates

Oxo-acids by 2,4 dinitrophenyl-hydrazones & appear yellow
Amino acids by ninhydrin & appear purple

Question 7

What are the 6 steps involved in the experiment?

what tubes are 5 and 9 and 10?

ninhydrin sprays:

If tube 1 contains pyruvate, buffer, heart extract & ethanol, what colour will appear? Why?
If tube 2 contains glutamate, buffer & extract, what colour will appear? Why?
If tube 3 contains pyruvate, glutamate, & extract, what colour will appear?
If tube 4 contains glutamate, NAD+ and extract, what colour will appear?

Answer 7

1. Add reagents to 10 tubes
2. in tubes 1-4 add 1.5ml ethanol
3. in tubes 5-10 add 1.5 ml 2,4 dinitrophenylhydrazine solution.
4. mix & allow stand & put 5-10 mins in 37 degrees water bath & then add 0.5ml ethyl acetate (to extract 1.5 ml dinitrophenylhydrazine)
5. centrifuge all 10 tubes & remove precipitated protein & ethyl acetate (5-10)
6. spot samples on TLC plate

5 = standard 2,4 dinitrophenylhydrazine of pyruvate (yellow)
9 = standard 2-oxoglutarate (yellow)
10 = 2,4 dinitrophenylhydrazine solution

no spot- no transamination or deamination

purple spot- no transamination/deamination but amino acid glutamate present

2 purple spots- glutamate and alanine (transamination)

1 purple spot- deamination occurs but can’t visualise oxo acid, but glutamate is present

Question 8

2,4-dinitrophenylhydrazine stain

if tube 5 contains pyruvate, buffer & extract, what spots appear?

if tube 6 contains glutamate, buffer & extract, what spots appear?

if tube 7 contains pyruvate, glutamate & extract, what spots appear?

tube 8 contains glutamate, NAD+ & extract, what spots appear?

if 9 contains 2 oxoglutarate, buffer & extract, what spots are there?

10 contains buffer & extract buffer, what spots are there?

Answer 8

2 yellow spots- pyruvate (2 isomers)

no spot (no transamination/deamination) except for where 2-4dinitrophenylhydrazine migrates to

2-3 yellow spots- (2) pyruvate and 2-oxoglutarate

1 yellow spot = 2-oxoglutarate

1 yellow spot for 2-oxoglutarate

none except for when 2-4DNPH migrates

Question 9

what is the TLC stationary phase?

what are the solvent systems for amino acids & oxo acids?

why do you have to keep the plates in a fume cupboard?

Answer 9

silica gel

ethanol: aqueous ammonia (conc) 70:30 (amino acids)
n-butanol: ethanol: aqueous ammonia (conc) 70:10:20 (oxoacids)

ammonia