Experiment B: transamination Flashcards
what happens to excess amino acids in the body?
- amino acids cannot be stored
- must be deaminated
- carbon skeletons used as metabolic fuel
- or stored as glycogen or fats
conversions into which amino acids are particularly important?
- into glutamate for oxidative deamination, releasing an ammonium ion
- into aspartate, which may contribute its amino group directly in urea synthesis
what are the group of enzymes called that move amino groups between amino acids and oxoacids?
transaminases
what is the enzyme called that catalyses the oxidative deamination of glutamate?
glutamate dehydrogenase
what is transamination?
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
give an example of a transamination reaction
pyruvate + glutamate —> alanine + α-ketoglutarate
what is the tightly bound cofactor of transaminases?
pyridoxal phosphate (derived from vitamin B6)
what is the equilibrium position of reactions catalysed by transaminases?
K(eq) is close to 1, suggesting that the equilibrium position is in the centre and that the reaction is freely reversible
which coenzymes are used by glutamate dehydrogenase?
either NAD+ or NADP+
give an example of oxidative deamination
glutamate + NAD+ + H2O —> α-ketoglutarate + NADH + H+ + NH4+
what is oxidative deamination?
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
what happens to the ammonium ion produced by oxidative deamination?
- transported into the liver as glutamine
- it is then converted into carbamoyl phosphate, which enters the urea cycle
what role does water play in oxidative deamination?
it is the hydrolysing agent
how is the heart extract prepared?
- minced
- homogenised in ice-cold buffer
- centrifuged at 20 000 g for 20 minutes
- supernatant is dialysed exhaustively against ice-cold buffer
what buffer is used in the exhaustive dialysis of the heart extract?
0.05M phosphate at pH 7.4
which substances are yellow?
the 2,4-dinitrophenyl-hydrazones of the oxoacids
how are the amino acids detected?
by spraying with ninhydrin and heating to give a purple colour
outline the procedure used in the experiment
- 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
why are ethanol and 2,4-DNP added?
to stop the reaction
why is ethyl acetate added to tubes containing 2,4-DNP?
it extracts the 2,4-dinitrophenyl-hydrazones
why are the tubes centrifuged?
to separate the ethyl acetate and to remove the precipitated protein
which solvents are used for TLC?
for amino acids, ethanol:aqueous ammonia (conc) 70:30
for oxoacids, n-butanol:ethanol:aqueous ammonia (conc) 70:10:20
what is the source of the transaminases and glutamate dehydrogenase that facilitate the experiment?
the heart muscle
what occurs in each of the 10 tubes?
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
explain the mechanism of ninhydrin
- 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
what is an aminopeptidase?
catalyses the cleavage of amino acids from the amino terminus
what is a carboxypeptidase?
catalyses the cleavage of amino acids from the carboxyl terminus
explain the mechanism of 2,4-dinitrophenolhydrazine
- 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
what is another name for 2-oxoglutarate?
α-ketoglutarate
