Overview

It is the polymeric nitrogen containing compounds proteins and nucleic acids that define the major attributes of organism such as function and structure. Operation and mechanism of metabolic pathways is provided by proteins. Genetic information is stored in nucleic acid polymers. Each of the monomer of these macromolecules has an individual metabolic pathway. In addition, the monomeric nucleotides are essential for energy turnover as key intermediates in all metabolic pathways and also as second messenger molecules, often in form of cyclic nucleotides.

Amino acids contribute to carbohydrate synthesis via gluconeogenesis, to fat synthesis or energy production via acetyl-CoA, and special nitrogen compounds such as catecholamines (neurotransmitters), thyroid hormones, creatine(-phosphate), the protoporphyrin ring (heme), and contribute to nucleic acid and phospholipid synthesis as nitrogen group donor.

Microbial nitrogen fixation
(KEGG pathway MAP00910)

All nitrogen metabolism is based on a recycling of ammonia NH₃ in its neutral or charged form NH₄⁺(ammonium ion). Ammonia, however, is not a major form of nitrogen on earth, instead it has to be replenished to support a growing need of life forms. As simple as it may sound, a growing bacterial culture needs raw material in form of small organic and inorganic molecules. NH₃ ultimately is derived from atmospheric N₂. In a process called nitrogen fixation, some bacterial species, the symbiotic eubacteria Rhizobium (in plant root nodules) and the archaea cyanobacteria (formerly blue-green algae) contain an enzyme complex for the reduction of molecular nitrogen to ammonia. This is the nitrogenase complex and contains Fe-S and Mo-Fe cofactors for the transfer of electrons from ferredoxin to N₂. The process of nitrogen reduction is extremely energy dependent. The triple bond energy in molecular nitrogen is 225kcal/mol and the industrial production of ammonia requires temperatures of 500 degrees Celsius and a pressure of 300 atmospheres. Rhizobium uses 8 reducing equivalents and 16 ATPs:

             N₂ + 8e^- + 8H⁺  + 16ATP + 16H₂O = 2NH₃ + H₂ + 16ADP + 16P_i + 8H⁺

This reaction is catalyzed by the hetero-oligomeric protein complex composed of a reductase and a nitrogenase part. The reductase is a homodimer containing a 4Fe-4S cluster and an ATP binding site at the subunit interface. The nitrogenase (EC 1.18.6.1; PDB entry 1N2C) is a hetero-tetramer with a subunit stoichiometry of a2b2. The ab interface contains the so called P cluster (containing two 4Fe-4S clusters) which oxidizes the reductase and is oxidized by the Mo-Fe cofactor which contains two Mo-3Fe-3S clusters comprising the N2 binding site. For a complete structure of  nitrogenase complex from Azotobacter Vinelandii click here.

The reductase (PDB structure from Clostridium Pasterianum; EC 1.19.6.1) contains an 4Fe-4S complex used to oxidize ferredoxin, which is supplied either by photosynthetic membranes (PSI) or from oxidative catabolism. The reductase donates 8 electrons in succession to the nitrogenase cofactor, a molybdenum-iron containing active center, where one molecule of N₂ is reduced in the presence of protons to 2 NH₃, and H₂ as a byproduct. The reduction catalysis is powered by sixteen ATP molecules hydrolyzed by the reductase subunit. Molecular oxygen is a strong inhibitor of the nitrogenase Mo-Fe cofactor and is removed by the plant oxygen binding protein leghemoglobin in the root nodules.

Dietary nitrogen

The majority of useful nitrogen for animal metabolism comes from proteins in the form of reusable ammonia (NH3). Nitrogen is fixated in form of ammonia by microorganisms (see chapter on amino acid synthesis) and all 'higher' forms of life (eukaryotes) depend on this primordial source of nitrogen extracted from the air. The 'usefulness' of proteins depends on four distinct properties:

1. total amount of protein ingested
2. digestibility of proteins
3. amino acid composition of proteins
4. total caloric intake

Essential amino acids

Some amino acids may be synthesized in human cells, some however cannot. The latter are referred to as essential amino acids meaning that they are required dietary components. The non-essential amino acids, however, can be interconverted into each other or synthesized de novo from carbohydrate, nucleic acid, or lipid intermediates, provided that an adequate source of total nitrogen is available. For essential amino acids there is no metabolic pathway for de novo synthesis except in bacteria and plants. Humans therefore need a daily balanced intake of those essential amino acids. Meat and milk provide such a balanced amino acid diet by virtue of the evolutionary relationship with between animals and humans. The list of the essential amino acids for human protein synthesis includes the branched amino acids Isoleucine, Leucine, and Valine, the sulfur containing Methionine, the hydrophilic amino acids Lysine and Threonine, and the aromatic amino acids Phenylalanine and Tryptophan. The amino acids Arginine and Histidine are synthesized in human cells, but only slowly and thus can be considered essential, if they become rate limiting factors for protein synthesis.

Nitrogen balance

The nitrogen balance is an indication of protein synthesis and degradation. A positive nitrogen balance indicates that the intake of nitrogen containing compound exceeds the nitrogen lost from the body. A positive nitrogen balance correlates with a net synthesis of proteins and nucleic acids. This obviously describes a state of growth of an organism  -  childhood, pregnancy, recovering from illness. The opposite results in a net degradation of proteins. Less nitrogen is taken up than is lost, is a state of negative nitrogen balance. The omission of essential amino acids, and this needs to affect only one type, results in a negative nitrogen balance, since it will be rate limiting for protein synthesis. A healthy body is characterized by a nitrogen equilibrium (steady state equilibrium) where intake and loss of nitrogen are equal. Here is a quote from Murray's "Essentials of human metabolism":

" In a way this equilibrium description satisfies our common sense observation of non growing adults. The interpretation would be that the body uses only as much protein nitrogen from the diet as necessary to replace digestive enzymes, gastrointestinal cells (GI) lost in the feces, or any degenerated tissue components, such as skin cells or erythrocytes that wear out during normal use. ... Why then does the body have such high requirements specifically for protein as well as for essential amino acids? Why could these energy requirements not be met almost entirely by increased carbohydrate and lipid intakes? The answer to these questions requires a different concept of metabolism than is implied by the terms 'chemical equilibrium' or the replacement of components due to wear and tear."

The key regulatory element of this turn-over process is the nitrogen balance reflected as the free amino acid pool. This pool is regenerated by dietary proteins and tissue protein degradation and is the source of protein synthesis as well as nitrogen secretion while maintaining nitrogen level homeostasis.

Turnover rates are best described as biological half-life time. An estimated 2 to 3 weeks has been given for a complete turnover of all body proteins (with a considerable variation). The turnover rate of individual proteins or specific families of proteins may be less than an hour. In actual numbers the rate of protein synthesis every day is estimated at about 500g or nearly five times the average dietary intake. There is obviously a highly efficient amino acid recycling machinery at work. This, in short, is the significance of amino acid metabolism.

Liver nitrogen metabolism

The liver is the main metabolic organ utilizing amino acids for tissue protein synthesis, heme formation, pyrimidine and purine synthesis (nucleotide precursors), ketone body and carbohydrate formation, de novo synthesis of non-essential amino acids, and finally excrete surplus nitrogen via the urea cycle. The liver thus is the gatekeeper of the nitrogen balance in animals, its intake and excretion. Because of its central role of regulating and coordinating body metabolism, protein turnover in liver is particularly fast. This ensures a reliable supply of (intact) blood plasma proteins, and liver resident proteins obviously affect liver metabolism which affects all other tissues, too. Finally, some proteins may be rapidly degraded to provide a constant level of free amino acids for the formation of ketone bodies, carbohydrates, nucleic acids, and heme. Hormonal control (glucocorticoids) makes sure that a starving body first breaks down proteins from non-essential organs like skeletal muscle, while liver enzymes for gluconeogenesis and urea cycle (nitrogen decontamination of the body) are enhanced. The liver acts as an aminostat. Free amino acid levels in blood plasma as well as plasma proteins are maintained at constant levels despite fluctuations in intake and tissue demand.

Glutamate (C00025) and glutamine (C00064) are the two important amino acids in recycling ammonia in our body instead of excreting it as waste in form of urea (C00086). Glutamine is synthesized from glutamate by incorporation of an NH3 into the carboxyl group forming an amide. This step requires ATP and is catalyzed by glutamine synthetase (EC 6.3.1.2). The coupling of glutamine synthesis with ATP hydrolysis renders the reaction irreversible. The back reaction  -   the regeneration of glutamate from glutamine   -    is catalyzed by glutaminase  (EC 3.5.1.2), which deaminates glutamine via a hydrolysis reaction. The concerted control of these two enzymes is responsible for the maintenance of the glutamine pool in blood. An example of controlling the NH3 levels is enhanced gluconeogenesis in specialized organs such as muscle and brain. Carbohydrates are synthesized from amino acid sources increasing the cellular ammonia levels. They are secreted by the peripheral tissues in form of glutamine (to avoid that nitrogen is excreted from the body) which is taken up by hepatocytes where the NH3 is re-used for amino acid and nucleotide synthesis.

Aminotransferases

Aminotransferases are a class of enzymes responsible to attach and remove amino groups from alpha-carbons of amino acids and keto acids. Aminotransferases (or transaminases) link amino acid metabolism with other pathways, most importantly the citric acid cycle. The reaction catalyzes the transfer from an alpha amino acid to an alpha keto acid. The transferase using alpha-ketoglutarate and alpha-glutamate as acceptor and donor group, respectively, takes a central role in the linkage between amino acid metabolism and citric acid cycle. This reaction is coupled with the enzyme glutamate dehydrogenase which catalyzes the amination-deamination equilibrium between alpha-ketoglutarate and glutamate. Thus, the interplay of the two enzymes glutamate transaminase (EC 2.6.1.1; transferase) and glutamate dehydrogenase (EC 1.4.1.2) is essential in the control of nitrogen balance in the body.

Aminotransferase reactions involve little change in free energy (they catalyze the reaction close at its chemical equilibrium) and the direction of the catalysis is essentially controlled by the concentration levels of the reactants. The dehydrogenase activity is controlled by the redox potential of the cell in form of NADH. The amination (NH3) reaction is coupled to a reduction step using NADH/H+ (oxidized) and alpha-ketoglutarate (reduced) while producing glutamate, NAD+ and water.

Aminotransferase reactions depend on vitamin B6, namely its derivative pyridoxal-phosphate (C00018), which acts as a coenzyme in the reaction, temporarily binding the transferred amino group. The pyridoxal phosphate group converts to pyridoxamine phosphate during the catalysis. Pyridoxal phosphate, however, is quite a versatile coenzyme being used in enzymes catalyzing the following reactions by temporarily accepting the transferred reactant (hint: click on the link above to pyridoxal phosphate and explore the long list of enzymes (117) that use this functional group):

- transamination
- decarboxylation
- deamination
- racemization
- aldole cleavage
- elimination and replacement reactions at b carbons and g carbons

is catalyzed in two steps. First by glutamate dehydrogenase:

followed by alanine-glutamate aminotransferase:

All steps are reversible. The dehydrogenase reaction occurs in the mitochondrial matrix where it directly interacts with NAD+ and alpha-ketoglutarate. The dehydrogenase is under allosteric control of the energy charge of the cell. High levels of ATP and GTP inactivate the enzyme while high levels of ADP and GDP activate it.

Non-essential amino acids

All non-essential amino acids except for tyrosine and cysteine are derived and are dependent on transamination from glutamate. Proline, ornithine, arginine obtain their carbon units and amino nitrogen from glutamate. Alanine, serine, glycine obtain their C3 carbon units from glycolytic intermediates and the amino nitrogen from glutamate (Note: glycine is a C2 amino acid derived from serine by decarboxylation; see one carbon metabolism). Aspartate derives its carbon backbone from oxaloacetate (C4) and amino nitrogen from glutamate. In fact, glutamate-dehydrogenase in combination with any aminotransferase is capable of forming any non-essential amino acid, given the occurrence of the proper alpha-keto acid and a source for ammonia. This process is called reductive amination (see above formation of alanine from pyruvate). The main purpose of reductive amination is to recycle NH3 instead of excreting it in form of urea and to preserve other amino acids which could serve as amino group donor. 

Reductive amination is the first of three processes in liver for ammonia incorporation. The second important process is the formation of glutamine, which serves as a reservoir for ammonia for all organs and is maintained as blood glutamine levels by liver cells. Glutamine serves as transport mechanism of NH3 between organs. Third, liver can form carbamoyl-phosphate, which is necessary for the formation of pyrimidine bases of nucleotides and the production of urea via the enzymes of the urea cycle.

Urea cycle
(KEGG pathway MAP00220)

The urea cycle is a liver resident process removing nitrogen in form of ammonia to be excreted from the body. The cycle involves two amino acids which are not used for protein synthesis. These are ornithine and citrulline. Ornithine has a role analogous to that of oxaloacetate in the citric acid cycle. It provides the carbon backbone and works as a catalytic carrier, but is not itself used up in the cyclic reaction. 

Ornithine has a terminal amino group that serves as a hook or handle for the incoming carbamoyl phosphate (C00169), a small molecule formed from CO₂ and NH3 and ATP as phosphate donor. Carbamoyl-phosphate is catalyzed by carbamoyl-phosphate synthetase (EC 6.3.4.16; forming carbamoyl- phosphate) in the mitochondrial matrix and requires the hydrolysis of 2 molecules of ATP. Carbamoyl is transferred to the ornithine amino group driven by the hydrolysis of its phosphate ester bond. Citrulline (C00327) is the product of this reaction and will be transported, together with aspartate, out of the mitochondria and into the cytoplasm. There, aspartate and citrulline are combined into the metastable intermediate argininosuccinic acid (C03406) using one molecule of ATP as energy source. Argininosuccinate is cleaved into arginine and fumarate. The latter is recycled back into the mitochondria for use by the citric acid cycle, while most of the arginine is converted by arginase (EC 3.5.3.1) to urea (C00086) and ornithine, thus completing the cycle. Like ornithine-carbamoyl transferase, arginase is a liver specific enzyme (in the cytoplasm) and only in those animals (mammals) which convert their nitrogen waste to urea. Note that in muscle most arginine synthesized is instead used for protein synthesis and creatine (C00300) formation. The phosphorylated creatine-P is used as an intermediate energy storage device under anaerobic conditions in skeletal muscle.

Fumarate is used to regenerate aspartate used up by urea formation. This is done by funneling fumarate back into the citric acid cycle and removing it in form of oxaloacetate (alpha keto acid) which can equilibrate with aspartate (alpha amino acid) catalyzed by an amino transferase reaction. Both the Krebs and urea cycle are thus strictly interrelated with the former providing essential intermediates plus carbon dioxide and energy in form of ATP (from oxidative phosphorylation, strictly speaking).

The urea cycle is part of two cellular compartments, the mitochondrial matrix which performs the biosynthetic part of the precursors citrulline and aspartate, and the cytoplasm, which after formation of arginino-succinate cleaves this intermediate into three different products, one of which is the net product (urea), the other two (ornithine and fumarate) are recycled into the matrix compartment and their respective cycle to start a new round of urea formation

Ammonium ion metabolism

Nitrogen can also be excreted as ammonium. This process is controlled by the kidney and is used to control the blood plasma pH. The blood plasma pH, however, is determined by other factors as well, such as organic acids (amino acids) and carbonic acid (CO₂ levels). Ammonium metabolism in kidney functions to depose H⁺ in urine. In a first reaction, kidney enzymes deaminate glutamine in two steps to a-ketoglutarate. The first side chain deamination is catalyzed as simple hydrolysis and is not reversible.

This process is stimulated by inorganic phosphate. The free ammonia equilibrates with protons to ammonium:

                                        NH3 + H⁺  =  NH₄⁺

and is trapped in the charged form inside kidney cells. The non-electrolytic ammonia is freely diffusible across cell membranes. Glutamine is the nontoxic form of NH3 and shuttles it between liver and kidney in the blood plasma. The kidney functions as H+ sink and protons are disposed in form of NH₄⁺ while maintaining charge homeostasis using phosphate or acetoacetate.

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