Experiment B: transamination

Question 1

what happens to excess amino acids in the body?

Answer 1

• amino acids cannot be stored
• must be deaminated
• carbon skeletons used as metabolic fuel
• or stored as glycogen or fats

Question 2

conversions into which amino acids are particularly important?

Answer 2

• into glutamate for oxidative deamination, releasing an ammonium ion
• into aspartate, which may contribute its amino group directly in urea synthesis

Question 3

what are the group of enzymes called that move amino groups between amino acids and oxoacids?

Answer 3

transaminases

Question 4

what is the enzyme called that catalyses the oxidative deamination of glutamate?

Answer 4

glutamate dehydrogenase

Question 5

what is transamination?

Answer 5

the transfer of an amino group from an α-amino acid to a 2-oxo acid (or α-keto acid) to produce a new amino acid and a new α-keto acid

Question 6

give an example of a transamination reaction

Answer 6

pyruvate + glutamate —> alanine + α-ketoglutarate

Question 7

what is the tightly bound cofactor of transaminases?

Answer 7

pyridoxal phosphate (derived from vitamin B6)

Question 8

what is the equilibrium position of reactions catalysed by transaminases?

Answer 8

K(eq) is close to 1, suggesting that the equilibrium position is in the centre and that the reaction is freely reversible

Question 9

which coenzymes are used by glutamate dehydrogenase?

Answer 9

either NAD+ or NADP+

Question 10

give an example of oxidative deamination

Answer 10

glutamate + NAD+ + H2O —> α-ketoglutarate + NADH + H+ + NH4+

Question 11

what is oxidative deamination?

Answer 11

the conversion of an amino acid into the corresponding keto acid by replacing the amine group with a ketone group; the amine is lost as ammonia/ammonium ion

Question 12

what happens to the ammonium ion produced by oxidative deamination?

Answer 12

• transported into the liver as glutamine

- it is then converted into carbamoyl phosphate, which enters the urea cycle

Question 13

what role does water play in oxidative deamination?

Answer 13

it is the hydrolysing agent

Question 14

how is the heart extract prepared?

Answer 14

• minced
• homogenised in ice-cold buffer
• centrifuged at 20 000 g for 20 minutes
• supernatant is dialysed exhaustively against ice-cold buffer

Question 15

what buffer is used in the exhaustive dialysis of the heart extract?

Answer 15

0.05M phosphate at pH 7.4

Question 16

which substances are yellow?

Answer 16

the 2,4-dinitrophenyl-hydrazones of the oxoacids

Question 17

how are the amino acids detected?

Answer 17

by spraying with ninhydrin and heating to give a purple colour

Question 18

outline the procedure used in the experiment

Answer 18

• incubate all tubes at 37C for 30 minutes
• add ethanol/2,4-dinitrophenol
• incubate those test tubes containing 2,4-DNP for a further 5 minutes and add ethyl acetate
• centrifuge all tubes for 5 minutes at 3/4 speed

Question 19

why are ethanol and 2,4-DNP added?

Answer 19

to stop the reaction

Question 20

why is ethyl acetate added to tubes containing 2,4-DNP?

Answer 20

it extracts the 2,4-dinitrophenyl-hydrazones

Question 21

why are the tubes centrifuged?

Answer 21

to separate the ethyl acetate and to remove the precipitated protein

Question 22

which solvents are used for TLC?

Answer 22

for amino acids, ethanol:aqueous ammonia (conc) 70:30

for oxoacids, n-butanol:ethanol:aqueous ammonia (conc) 70:10:20

Question 23

what is the source of the transaminases and glutamate dehydrogenase that facilitate the experiment?

Answer 23

the heart muscle

Question 24

what occurs in each of the 10 tubes?

Answer 24

1&5 - control; pyruvate; no reaction
2&6 - control; glutamate; no reaction
3&7 - transamination; alanine and α-ketoglutarate
4&8 - pxidative deamination; α-ketoglutarate
9 - control; α-ketoglutarate; no reaction
10 - control; no reaction

Question 25

explain the mechanism of ninhydrin

Answer 25

• reacts with the amino group of the free amino acid
• which undergoes further decarboxylation and removal of an aldehyde
• to produce an adduct which forms a purple complex with another molecule of ninhydrin

Question 26

what is an aminopeptidase?

Answer 26

catalyses the cleavage of amino acids from the amino terminus

Question 27

what is a carboxypeptidase?

Answer 27

catalyses the cleavage of amino acids from the carboxyl terminus

Question 28

explain the mechanism of 2,4-dinitrophenolhydrazine

Answer 28

• condensation reaction (loss of H2O)
  OR
• addition-elimination reaction - nucleophilic addition of -NH2 group to the C=O group followed by the removal of H2O
• results in a yellow/orange precipitate
• crystals of different hydrazones have different melting/boiling points
• the identity of the substrate can be identified by derivatisation

Question 29

what is another name for 2-oxoglutarate?

Answer 29

α-ketoglutarate