Metabolism on-line - the virtual tutorial room

 

copyright © 2008 - 2014 David A Bender

Transamination and deamination of amino acids

The first step in the catabolism of most amino acids is removal of the amino group to form the alpha-keto-acid (correctly an oxo-acid), which is the carbon skeleton of the amino acid.

A small number of amino acids undergo oxidative or non-oxidative deamination. For example, glutamate is oxidised to alpha-ketoglutarate by glutamate dehydrogenase, glycine is oxidised to glyoxylate by glycine oxidase. There is also a general amino acid oxidase, but this has very low activity, and is not of great importance in amino acid metabolism. Serine undergoes non-oxidative deamination to pyruvate, catalysed by serine deaminase.

For other amino acids there is no direct deamination, but they can undergo transamination. This is a reaction between an amino acid and a keto-acid in which the amino group is transferred from the donor amino acid onto the acceptor keto-acid , leaving the carbon skeleton (keto-acid) of the donor amino acid and forming the amino acid corresponding to the acceptor keto-acid.

In the first half-reaction, the amino group is transferred from the substrate amino acid onto the prosthetic group, pyridoxal phosphate, releasing the keto-acid and forming pyridoxamine phosphate at the active site. In the second half reaction the amino group is transferred onto the acceptor keto-acid, forming the product amino acid, leaving pyridoxal phosphate at the active site, ready to undergo another reaction cycle.

 

 

 

 

 

 

 

Commonly, the acceptor keto-acid is either alpha-ketoglutarate (forming glutamate) or oxaloacetate, forming aspartate.

 

How can transamination linked to alpha-ketoglutarate (forming glutamate) account for the overall deamination of most amino acids?

A simple two reaction pathway involving transamination to form glutamate and glutamate dehydrogenase to release the ammonium and reform alpha-ketoglutarate will allow overall deamination of most amino acids for which there is an alpha-ketoglutarate-linked transaminase.

Aspartate transaminase catalyses a reaction between aspartate and alpha-ketoglutarate to form oxaloacetate and glutamate.

 

 

 

 

 

 

How can transamination linked to oxaloacetate (forming aspartate) account for the overall deamination of most amino acids?

Now we need three reactions:

transamination linked to oxaloacetate, forming aspartate,
aspartate transaminase, forming glutamate from alpha-ketoglutarate and reforming oxaloacetate,
glutamate dehydrogenase to release the ammonium and reform alpha-ketoglutarate.

If the keto-acid corresponding to an amino acid can be synthesised from a common metabolic intermediate, and not only from the amino acid itself, can you explain how the non-essential amino acids are synthesised?

As well as being a mechanism for catabolism of amino acids, transamination also provides a mechanism for synthesis of those amino acids whose carbon skeletons are intermediates in carbohydrate metabolism - the non-essential amino acids.

The treatment of patients in renal failure involves feeding a low protein diet, in order to minimise the burden of nitrogen to be excreted (mainly as urea), while providing just enough protein to meet requirements.

Why do you think that providing the keto-acids of essential amino acids is beneficial in such cases?

If the keto-acids of essential amino acids are provided in the diet then they are substrates for transamination to yield the amino acids, so using nitrogen that would otherwise be metabolised to ammonium and then urea. This permits a greater reduction in total protein intake and further reduces the burden of nitrogen to be excreted.

Experiments with transaminases

In these experiments you will investigate the activities of two transaminases:

aspartate transaminase, which catalyses the transfer of the amino group from aspartate, forming oxaloacetate, onto alpha-ketoglutarate, forming glutamic acid.

alanine transaminase, which catalyses the transfer of the amino group from alanine, forming pyruvate, onto alpha-ketoglutarate, forming glutamic acid

 

Experiment: transamination and deamination of glutamate by heart muscle

Ox heart muscle was homogenised in ice cold buffer and centrifuged at 20,000 g for 20 minutes to remove intact cells, cell debris, nuclei and mitochondria. The resultant supernatant was dialysed against three changes of 0.05 mol /L phosphate buffer at pH 7.4.

What is dialysis, and why was the supernatant dialysed before setting up the incubations?

In dialysis the sample is placed in a sac of semi-permeable membrane with pores that will permit small molecules, but not proteins, to diffuse across, and equilibrate in the larger volume of buffer outside. After three changes of buffer on the outside, the concentration of small molecules in the sample is very low.

The supernatant was dialysed to remove amino acids, keto-acids and other substrates that were present in the original tissue sample, so that only those substrates that are added in the incubations will be available for metabolism. It would be impossible to interpret the results if there were substrates present form the original heart muscle preparation.

After incubation of the heart preparation with substrates and cofactors, amino acids can be detected on thin-layer chromatograms by reacting them with ninhydrin; after heating the amino acids show up as purple spots. The chromatograms are developed in solvent (ethanol : ammonium hydroxide 70 : 30) then sprayed with ninhydrin solution and heated.

Keto-acids such as alpha-ketoglutarate and pyruvate can be detected by reacting them with dinitrophenylhydrazine, to form coloured dinitrophenylhydrazones, then separating them by thin-layer chromatography. In this case the solvent is n-butanol : ethanol : ammonium hydroxide 70 : 10 : 30).

Ten incubations were set up, as shown in the table below. All solutions were prepared in 0.05 mol /L sodium phosphate buffer at pH 7.4. The heart extract was added last, after all the other reagents have been added, then the contents of each tube were mixed, and they were placed in a water bath at 37°C for 30 min.

 

At the end of the incubation, the reactions were stopped as follows:

tubes 1 - 4: addition of 1.5 mL ethanol
tubes 5 - 10: addition of 1.5 mL dinitrophenylhydrazine solution

After allowing the tubes to stand for a few minutes, 0.5 mL ethyl acetate was added to tubes 5 - 10 and they were shaken well; this extracts the dinitrophenylhydrazones of the keto-acids into the ethyl acetate.

The tubes were centrifuged to remove the precipitated protein and to separate the ethyl acetate in tubes 5 - 10.

1 mL samples of the reaction mixtures were spotted onto two separate thin-layer chromatography silica gel plates.

The 10 incubations were as shown below - all volumes are in mL.

	1	2	3	4	5	6	7	8	9	10
0.1 mol /L sodium pyruvate	0.5	-	0.5	-	0.5	-	0.5	-	-	-
0.1 mol /L sodium glutamate	-	0.5	0.5	0.5	-	0.5	0.5	0.5	-	-
0.005 mol /L NAD+	-	-	-	0.5	-	-	-	0.5	-	-
0.1 mol /L sodium alpha-ketoglutarate	-	-	-	-	-	-	-	-	0.5	-
phosphate buffer	0.5	0.5	-	-	0.5	0.5	-	-	0.5	1.0
heart extract	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5

 

What is the purpose of incubation no 10?

See the answer