Transamination and deamination of 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.

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

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.

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.

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.

This is a control, with no added substrate, to check that there were no substrates remaining in the dialysed heart preparation.

Plate 1 shows the amino acids at the end of the incubation, stained with ninhydrin.

The samples are:

1) 0.2 mol /L alanine
2) incubation no 1
3) incubation no 2
4) incubation no 3
5) incubation no 4
6) 0.2 mol /L glutamic acid

Plate 2 shows the dinitrophenylhydrazones of the keto-acids at the end of the incubation, after extraction into ethyl acetate.

The samples are:

1) incubation no 5
2) incubation no 6
3) incubation no 7
4) incubation no 8
5) incubation no 9
6) incubation no 10

This is present in all six incubations, regardless of whether there was any keto-acid present. It is unreacted dinitrophenylhydrazine.

In plate 1, alanine has only been formed in incubation no 3 (channel 4). This must be the result of transamination between glutamate and pyruvate.

In plate 2, alpha-ketoglutarate has been formed in incubations 7 (channel 3) and 8 (channel 4).

Incubation no 7 contained pyruvate and glutamate, so this must be the result of transamination between glutamate and pyruvate.
incubation no 8 contained glutamate and NAD+, so this must be the result of glutamate dehydrogenase

Assessing vitamin B6 nutritional status

The prosthetic group of transaminases, pyridoxal phosphate, is the metabolically active form of vitamin B6. The apoenzyme, without pyridoxal phosphate, is catalytically inactive. Under normal conditions there is always a small amount of inactive apoenzyme in tissues, although most is present as the catalytically active holoenzyme, with pyridoxal phosphate bound.

Red blood cells contain a relatively large amount of aspartate transaminase.

The catalytically inactive apoenzyme can be activated by pre-incubation with pyridoxal phosphate before adding the substrates. In vitamin B6 deficiency there will be a greater proportion of apoenzyme present than when vitamin B6 status is adequate

To perform the assay, red cells are centrifuged down from a blood sample taken in a heparinised tube to prevent coagulation, washed in 0.15 mol/L sodium chloride, then lysed by suspending the red cells from 1 mL of blood in 0.5 mL of distilled water.

The activities of two samples of the lysate are then measured:

a) One sample of the lysate is incubated at 20C with 0.1 mol/L pyridoxal phosphate in 0.1 mol/L phosphate buffer (pH 7.4) for 15 min
b) The other sample is incubated at 20C with 0.1 mol/L phosphate buffer (pH 7.4) for 15 min

and the two samples are then used for measurement of aspartate transaminase activity.

The sample pre-incubated with pyridoxal phosphate will have a higher transaminase activity, since any apoenzyme present will have been activated.

The ratio of activity in sample a (with added pyridoxal phosphate) / activity in sample b (without pyridoxal phosphate) is known as the activation coefficient.

An activation coefficient of < 1.25 suggests adequate vitamin B6 status; a value > 1.5 suggests deficiency. Values between 1.25 - 1.5 suggest marginal status, but would not be classified as deficiency.

An activation coefficient < 1.0 is impossible, because if all the enzyme in the sample was present as holoenzyme there would be no increase in activity on pre-incubation with pyridoxal phosphate, but there would be no loss of activity either, so the lowest possible value for the activation coefficient is 1.0

Transaminase activities in plasma are measured as part of liver function tests (see the exercise on hyperammonaemic coma due to liver failure).

The most commonly used method for measurement of transaminase activities is based on measurement of the appearance of the keto-acid product, using a second enzyme that reduces the keto-acid as it is formed, at the expense of NADH (which is oxidised to NAD+). The utilisation of NADH is followed by measuring the decrease in absorbance at 340 nm (the peak wavelength for the absorbance spectrum of NADH).

For measurement of aspartate transaminase, the sample is incubated with aspartate and alpha-ketoglutarate; as oxaloacetate is formed from the aspartate, it is reduced by malate dehydrogenase.

For measurement of alanine transaminase, the sample is incubated with alanine and alpha-ketoglutarate; as pyruvate is formed, it is reduced by lactate dehydrogenase.

The pyridoxal phosphate added to activate the apoenzyme has a strong absorbance around 340 nm. In addition, the red cell lysate has a strong colour and will also absorb around 340 nm.

Red blood cells contain a high activity of transaminases, so haemolysis would lead to falsely very high results for apparent plasma transaminases.

One method of measuring aspartate transaminase in red cell lysates is based on the release of tritiated water from [2,3-tritiated]aspartate (i.e. aspartate labelled with the radioactive isotope of hydrogen (tritium, 3H) on carbons 2 and 3) when it is transaminated to oxaloacetate.

It is only the hydrogen from carbon-2 of aspartate that is released in transamination, but 2-tritiated aspartate is not available.

The reaction is started by the addition of 0.1 mL of a solution containing 0.1 mol /L alpha-ketoglutarate and 0.05 mol /L [2,3-3H]aspartate and allowed to continue for 30 min at 37°C.

At the end of the reaction time, the tubes are plunged into a boiling water bath for 1 min then diluted with 1 mL ice-cold distilled water. 1 mL of a suspension of MB-5113 mixed-bed ion exchange resin (i.e. a resin which will take up both anions and cations) is then added, the tubes are mixed for 30 min, then centrifuged to precipitate denatured protein and the ion exchange resin. 0.2 mL samples of the supernatant are then mixed with a scintillator, and used for determination of radioactivity.

The mixed bed ion-exchange resin will remove all charged molecules - it will remove all of the unmetabolised substrate and the oxo-acid product - the only radioactive compound remaining will be tritiated water, which is what we want to measure.

For more on transaminases see the exercise on primary hyperoxaluria and kidney stones

Key points from this exercise:

• Transaminases catalyse the transfer of the amino group of an amino acid onto a keto-acid, forming the corresponding amino acid. The donor amino acid becomes its corresponding keto-acid.
• The prosthetic group of transaminases is pyridoxal phosphate, the metabolically active form of vitamin B6. The amino group form the donor amino acid is transferred onto pyridoxal phosphate, forming pyridoxamine phosphate, and releasing the keto-acid corresponding to the donor amino acid. The amino group is then transferred form pyridoxamine phosphate onto the acceptor keto-acid, forming the corresponding amino acid and leaving pyridoxal phosphate at the active site of the enzyme to undergo a further cycle of reaction.
• Many transaminases are linked to alpha-ketoglutarate, forming glutamate. This glutamate can be reoxidised to alpha-ketoglutarate by glutamate dehydrogenase, releasing ammonium. This provides a pathway for the overall deamination of a wide variety of amino acids.
• Many transaminases are linked to oxaloacetate, forming aspartate. The amino group can then be transferred onto alpha-ketoglutarate, in the reaction catalysed by aspartate transaminase, forming glutamate, which is a substrate for glutamate dehydrogenase, and reforming oxaloacetate. This provides a pathway for the overall deamination of a wide variety of amino acids.
• The keto-acids of the non-essential amino acids are relatively common metabolic intermediates, and if they can be synthesised in adequate amounts they are substrates for transamination, permitting synthesis of the non-essential amino acids.
• If the keto-acids of essential amino acids are supplied then the corresponding amino acids can be synthesised by transamination. This is useful in the treatment of renal failure, since it permits reduction in the amount of amino nitrogen to be metabolised and excreted.
• Red blood cells contain relatively high activities of transaminases, a proportion of which is present as the catalytically inactive apo-enzyme. Vitamin B6 nutritional status can be assessed by measuring the increase in activity of red cell transaminases after pre-incubation with pyridoxal phosphate.