Metabolism
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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?
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
What is the spot in each channel that has run to the solvent front?
This is present in all six incubations, regardless of whether there was any keto-acid present. It is unreacted dinitrophenylhydrazine.
What conclusions can you draw from these results?
Which enzyme is active in each of the incubations, transaminase or dehydrogenase?
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
| enzyme active: | 1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
| transaminase | + |
+ |
||||||||
| dehydrogenase | + |
+ |
||||||||
| neither | + |
+ |
+ |
+ |
+ |
+ |
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.
How might you use measurement of red blood cell aspartate transaminase activity to assess vitamin B6 nutritional status?
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.
Why would an activation coefficient < 1.0 suggest poor laboratory technique?
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.
Why do you think this uv absorbance method at 340 nm cannot be used to measure transaminase activity coefficients in red blood cell lysates?