As well as making up approximately 78% of the Earth’s atmosphere, nitrogen is a vital component of amino acids and nucleic acids which make up proteins and DNA. Nitrogen metabolism is therefore essential to the function and structure of many molecules and for the survival of all living organisms.
Despite its abundance in the atmosphere, nitrogen is relatively inert and most organisms are unable to utilise nitrogen in its pure form. The fixation of nitrogen by nitrifying bacteria, such as cyanobacteria and Rhizobium bacteria (found in the root nodules of leguminous plants), converts atmospheric nitrogen (N2) to soil based nitrates and ammonia (NH3). Following nitrogen fixation, reduced nitrogen in the form of ammonium ions (NH4+) can be used by organisms such as bacteria and plants to make more complex nitrogen containing compounds and make its way up the food chain.
In humans, reduced nitrogen enters the body through nitrogen containing foods such as proteins in our diet. Digestion of dietary protein begins in the stomach, where the proenzyme pepsinogen is converted to active pepsin A and catalyzes the primary stage of proteolysis. However, the majority of proteolysis takes place in the duodenum, with the aid of pancreatic proteases. These proteases (serine protease and zinc peptidase) act as both endo- and exo-peptidases; aiding amino acid and peptide degradation. The amino acids and peptides produced are taken up by the mucosal wall enterocytes of the intestines. Amino acids are the building blocks of proteins and hence are essential to growth and repair of cells
Nitrogen cannot be stored in the body like carbohydrates and fats. The rapid degradation of proteins means that deficiencies of just one amino acid can quickly limit essential protein synthesis. In order to maintain a healthy homeostasis of essential proteins in the body a nitrogen balance must be maintained – whereby nitrogen intake matches nitrogen excretion. Growth requires a positive nitrogen balance - in which the amount of nitrogen ingested exceeds that excreted. Ill health can be caused by inadequate protein intake and/or deficiency of one or more essential amino acids – termed a negative nitrogen balance. Nutritionally, the dietary proteins obtained from plants tend to be harder to digest and less concentrated than those from animal sources. Plant-sourced proteins are also more likely to be deficient in lysine, methionine and tryptophan residues.
Essential amino acids:
Whilst humans are able to synthesise many of the twenty standard amino acids, there are nine so-called essential amino acids which cannot be easily synthesised due to their complex structure (highly branched carbon chains, ring systems or inclusion of sulphur), or are synthesised too slowly to meet demand. These essential amino acids (outlined in Figure 1) must be obtained through our diet.
There are four non-essential amino acids and seven conditionally non-essential amino acids which do not need to be obtained by diet under normal conditions. Providing there is an adequate total nitrogen supply, all non-essential amino acids can be produced by the inter-conversion of one amino acid structure to another, or synthesised transamination reactions involving the carbohydrate, nucleic acid or lipid intermediates of major metabolic pathways.
Figure 1: The 20 standard amino acids, divided into essential, conditionally non-essential and non-essential categories; Non-essential amino acids are synthesised in the body, whilst essential amino acids must be obtained from food.
Transamination reactions are controlled by enzymes called aminotransferases. These enzymes are responsible for the removal and attachment of amino groups to the α-carbons of amino acids and keto acids, with the aid of a vitamin B6 derived pyridoxal-phosphate coenzyme. Aminotransferases link amino acid metabolism to other pathways such as the citric acid (TCA) cycle. The critical enzyme in this amino acid metabolism- TCA cycle linkage is glutamate transaminase; an aminotransferase which involves α-ketoglutarate as an amino acceptor and α -glutamate as an amino group donor. Together with glutamate transaminase, glutamate dehydrogenase maintains the amination-deamination balance between α-ketoglutarate and glutamate – an equilibrium that is fundamental to nitrogen balance control within the body.
Glutamate is a major component of many aminotransferase reactions – including the synthesis of aspartate and alanine. The basic synthesis pathways of these non-essential amino acids are outlined in Figures 2 & 3 below.
High serum levels of aspartate aminotransferase (AST) and alanine transaminase (ALT), can be used medically as indicators of tissue damage. ALT is fundamental in a process known as the glucose-alanine cycle.
Figure 2: Aspartate synthesis: illustrating the transfer of an amino group from the amino acid glutamate to oxaloacetate (a TCA cycle intermediate) by aspartate aminotransferase (AST), forming asparate and α-ketoglutarate.
Figure 3: Alanine synthesis: illustrating the transfer of an amino group from the amino acid glutamate to pyruvate (the end product of glycolysis) by alanine transaminase (ALT), forming alanine and α-ketoglutarate.
The glucose-alanine cycle:
The principle role of the glucose- alanine cycle (illustrated in Figure 4) is to allow skeletal muscle to eliminate nitrogen whilst simultaneously replenishing its energy supply. Glycolysis leads to production of pyruvate which is converted to alanine by ALT. Additionally, during periods of fasting, skeletal muscle protein undergoes degradation to provide fuel for respiration. Following muscle protein degradation, the abundant amino acid alanine enters circulation and is transported to the liver. In the liver, alanine is converted back to pyruvate and used in gluconeogenesis – providing glucose to fuel the skeletal muscle. The alanine which is transported from the muscle to the liver is converted to urea and excreted in urine as a product of the Urea Cycle – the principle method of nitrogen excretion.
Figure 4: The glucose-alanine cycle illustrating the simultaneous activities of the liver and the muscle in the metabolism of glucose and alanine.
Regulation of nitrogen metabolism by feedback inhibition:
The oxidative deamination of glutamate by glutamate dehydrogenase produces ammonia (NH3) and α-ketoglutarate. Ammonia can be highly neurotoxic at high concentrations; it can also cause severe liver dysfunction and disruption of the Urea Cycle. In order to regulate ammonia levels, elevated α-ketoglutarate concentrations are used to trigger the activation of glutamine synthetase. Glutamine synthetase acts as a catalyst the synthesis of glutamine, an important amino group donor in various reactions.
The activity of glutamine synthetase is controlled by nine allosteric feedback inhibitors. Six of these feedback inhibitors (histidine, tryptophan, carbomoyl phosphate, glucosamine-6-phosphate, AMP and CTP) are the products of pathways involving glutamine. The remaining three feedback inhibitors are alanine, serine and glycine – indicators of cellular nitrogen levels. Glutamine synthetase is also regulated by the adenylylation of tyrosine residues which results in increased sensitivity to the nine feedback inhibitors. Due to the high number of feedback inhibitors involved in the control of glutamine synthetase, the regulation of cellular nitrogen levels is extremely sensitive and should prevent dangerous accumulation of ammonia.
Ammonia produced by the urea cycle is highly toxic and must be removed from the organism's body through excretion. The form in which it is excreted largely depends on the availability of water. In fish ammonia is secreted directly into the surrounding water in a very dilute form. This is advantageous as no metabolic energy is expended processing ammonia into other molecules. In mammals and amphibians which normally do not have unlimited access to water, ammonia is first processed to urea. This less toxic nitrogen containing substance can then be excreted with far less water loss enabling the animal to conserve. For birds it is essential to minimize unnecessary body weight in order to facilitate flight. They convert ammonia to uric acid, which has very low solubility. This means very little water is needed to excrete waste nitrogen so the water stores they carry do not need to be as high.
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