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18.3: Nitrogen Excretion and the Urea Cycle

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    15033
  • The Urea Cycle

    Now we are in a position to see what happens to excess NH3/NH4+ that accumulates in the liver mitochondria. The ammonia that is formed in the liver through oxidative deamination by glutamate dehydrogenase, ends up in urea. Alternatively it could be added to glutamic acid to form glutamine which can head to the kidney where, after sequential reactions by glutaminase (a deamidation reaction) and glutamate dehydrogenase , it form ammonia for direct excretion in the urine. If your diet is high in proteins and hence amino acid, since they can't be stored as proteins per se, they are metabolized for energy and the metabolic products can be converted to fat or carbohydrate. The leaves excess nitrogen which ends up mostly as urea for excretion.

    Urea is a very water soluble, nontoxic molecule that biochemists use in the lab to chemically denature protein (at concentrations of 3-6 M). Clinical blood urea concentrations are expressed as Blood Urea Nitrogen (BUN) which range from 7 to 20 mg/dL N, which is equivalent to 2.5 to 7.1 mM urea. Serum levels depend predominately on the balance between urea's synthesis in the liver and its elimination by the kidney. Normal cellular urea concentrations should be similar.

    Urea is produced by a pathway called the urea cycle, as shown in the figure below.

    ureacycleFINAL.pngarginosuccinate lyase

    The enzymes names are:

    • CPS1: carbamoyl phosphate synthase I
    • OTC: ornithine transcarbamoylase
    • ASS1: arginosuccinate synthase 1
    • ASL: arginosuccinase lysase (aka arginosuccinase)
    • ARG1: arginase

    It is color coded to designate the sources of the C and N atoms. One N comes from ammonia (or indirectly from the amido N of glutamine), while the C atom comes from carbonate. The other comes from the amine of aspartate. Note that 3 ATPs are used to power the cycle. Also note the guanidino group of arginine (NH(NH2+)NH2) is the immediate donor of the 2 N atoms in the step that produces urea. Knowing that one amino acid was the immediate donor of the N atoms in urea, you would easily predict that.

    Now, let's look at some of the steps, starting with the ones that are powered by ATP, which are also the ones where nitrogen is incorporated into the precursors of urea.

    Carbamoyl phosphate synthase I (CPS I)

    This enzyme catalyzes the first "committed" path of the pathway and makes the reactant that enters the cycle, carbamoyl phosphate. It requires a specific cofactor, N-acetyl-glutamate (NAG), produced by the acetylation of glutamate by acetyl-COA using an enzyme NAG synthase which is activated by arginine. The reaction is not part of the cycle but provides the input for it.

    A cytosolic version of this enzyme, CPS II, is used to synthesize arginine and pyrimidine nucleotides using glutamine as a donor of NH4+which reacts with carboxyphosphate to produce carbamoyl phosphate. It is not regulated by NAG.

    CPS I provides a way to form a (C=O)NH2 unit for urea by condensing bicarbonate and ammonia to form a carbamate, which contains a high energy "motif" with respect to its hydrolysis produce. (Note: this does not imply that the broken bond is high energy, a misnomer found in many books.) Hence the reaction is powered by 2 ATPs as an energy source. Urea is a molecule then that "carries" both NH3/NH4+ but also carbonate.

    A reaction mechanism for carbamoyl phosphate synthase is shown below.

    CPS1.png

    The two high energy motif molecules (again with respect to their hydrolysis products) are protected from nonspecific hydrolysis as the mixed anhydride pass along a sequestered tunnel to more distal phosphorylation site in the multimeric enzyme.

    The structure below shows 2 ADP and a NAG bound to one chain of the multimer. The "free floating" NAG, ADP, and ions (magnesium and potassium) would be bound to the other monomer which is not shown.

    https://structure.ncbi.nlm.nih.gov/i...YEVatg25wW2U58

    iCn3D model: replace

    The protein exists in a monomer-dimer equilibrium mixture. Each monomer has 1 NAG and 2 ADP, which represent the two different binding site for the two different phosphorylation steps as shown in the reaction above. The two phosphorylation sites are connected by a tunnel allowing passage of the mixed anhydride to the carbamate phosphoryation site. The NAG is clearly an allosteric modifier as it is not bound near the phosphorylation sites. On binding to the apo form of the enzyme, it elicits a large conformation change allowing a competent enzyme conformation with a complete tunnel to predominate.

    Images of the tunnel for one monomer of the human CPS I enzyme are shown below.

    Top: ADPs are shown below in spacefill with CPK color. The tunnel between the two ADP sites is highlighted in magenta. The yellow molecule is NAG, which again exerts its activation effect by binding to an allosteric site, which effectively opens up the tunnel.

    5DOU_AChain_ChunnelAAs.png

    The next image shows the the enzyme monomer in a different orientation with a tunnel made using ChExVis: a tool for molecular channel extraction and visualization. The large red and green sphere shown the surface openings.

    5DOU_AChain_Chunnel_ChannelForRadiusKyteDoolittle.png

    The top panel in the figure below shows the radius in Angstroms of the tunnel going from the Red sphere to the Green sphere in the image above. The bottom panel shows a measure of the hydrophobicity (red)/hydrophilicity (blue) of the amino acids surrounding the tunnel.

    5DOU_AChain_Chunnel_RadiusKyteDoolittle.png

    ASS1: ArginoSuccinate Synthase 1

    Another ATP is cleaved to drive the conversion of citrulline to arginosuccinate which is could easily be cleaved in the next step to form the amino acid arginine.

    ASS1.png

    ASL: arginosuccinase lysase (aka arginosuccinase)

    This enzyme catalyses a beta-elimination reaction, which may proceed through either an E2 or E1 mechanism. A general but very abbreviated mechanism showing a two step (E1) elimination proceeding through a carbanion intermediate is shown below.

    ASL.png

    The model below arginosuccinate (AS11) bound in the active site of one of four monomers of ASL from Mycobacterium tuberculosis (6IEN). The key catalytic residues appear to be Ser 262 and His 161 which act as general acids/bases to facilitate proton abstraction and redonationd. Lys 288 appears to have a atypical pKa (6.0) which might faciliate deprotonation of Ser 262, allowing it to act as a general base. https://iubmb.onlinelibrary.wiley.com/doi/full/10.1002/iub.2000

    https://structure.ncbi.nlm.nih.gov/i...qKVpmzdwBzFJL8 O

    Ornithine Transcarbamyolase

    The reaction mechanism here is quite simple. The carbamoyl phosphate is a activated electrophile in which the carbonyl C is attacked by a deprotonated amino of ornithine to produce citrulline. The gene is found on the X chromosome in humans so mutations in males lead to a "hyperammonemic coma" and death. Heterozygous females can be asymptomatic or that generally proves to be fatal. Heterozygous females are either asymptomatic or have problems arising from defects in pyrmidine biosynthesis which can be alleviated by a low protein diet with addition of arginine.

    Arginase

    There are two general variants of this enzyme, Arginase I is a cytosolic enzyme expressed in the liver and is involved in urea synthesis. Arginase II is a mitochondrial enzyme and is involved more generally in arginine metabolism.

    "The substrate L-arginine binds to the active site, but does not coordinate to the metal ion centres directly. The metal-bridging hydroxide then initiates a nucleophilic attack on the substrate's guanidinium group, which leads to the formation of a tetrahedral intermediate. Following a proton transfer to the leaving amino group mediated by Asp-128, the collapse of the tetrahedral intermediate yields products l-ornithine and urea. In the final step, to regenerate the active site and release the products, a water molecule enters to bridge the binuclear manganese cluster, causing the urea product to move to a terminal coordination site on manganese 1. The dissociation of the product facilitates ionisation of the metal-bridging water molecule to yield the catalytically active hydroxide ion. Proton transfer from metal-bridging water to bulk-solvent is mediated by the side chain of His-141."

    The model below shows the active site of human arginase I (3KV2) in complex with the inhibitor N(omega)-hydroxy-nor-L-arginine, in which one of the guanidino Ns is replaced with an OH. The two grey spheres are Mn ions.

    Here is another model shown an active site water (480) near the MN ions. Water molecules can act as ligands and bind transition state metal ions through a coordinate covalent bond. The pKa of the bound water shifts to a lower value, making it more likely to deprotonate to form OH-, which is both a better base and nucleophile. Water 480 in the active site appears to form a coordinate covalent bond to the Mn ion forming an active site hydroxide which attacks the guanidino group of arginine, leading to the formation of urea and ornithine.

    ​A static image:

    3kuv_ActFinal.png

    ​Regulation

    If excess amino acids/proteins are consumed, change in gene expression can increase levels of the enzymes in the urea cycle. In the short term, the activity is regulated by the enzyme that provides access into the cycle, carbamoyl phosphate synthase, who activity is regulated by N-acetylglutamic acid and which catalyzes the rate-limiting step. As shown above, without the allosteric regulator, NAG, the enzyme is functionally inactive. The levels of NAG are determined by the enzyme NAG synthase, which itself is regulated by free arginine, the immediate precursor of urea in the cycle. Supplemental arginine is give patients with urea disorder system and its effect probably occurs through the regulation of NAG synthase.

    The link between the urea cycle and the TCA cycle

    Two of the major pathways you have explored, the TCA and urea cycles, are not linear but cyclic pathways. What advantages do "circular" offer over linear ones? Perhaps a comparison to economic pathways offers a clue. In a linear economy, raw materials are used to produce a product which is eventually discarded as trash. Unfortunately, linear pathways dominate our world at a great cost to our environment. To reduce the inefficiencies in a linear economy and make it more viable and to decrease environmental damage and associated climate change, the world should move to a circular economy. A circular economy is characterized by what has been referred to as the 3Rs: reduce, reuse and recycle. This saves resources and energy. These characteristics also apply to circular biochemical pathways as reduced levels of metabolites get reused and recycled.

    Given this, it should not come as a surprise that the urea and TCA cycle are linked, especially given that fumaric acid is a product of the urea cycle and a cyclic metabolite of the TCA cycle. In addition asparatic acid is one transaminatin step away for oxaloacetate.

    urea_TCA_Shuttle_Combo2.png

    One of the nitrogens in urea comes from the amine of aspartate, which can be formed through the transamination of oxalacetate from the TCA cycle to form aspartate. Since the TCA cycle is a cycle, an equivalent of one oxaloacetate must return to the cycle. It does so indirectly from fumarate, a TCA intermediate produced as a biproduct of the urea cycle, but the fumarate is produced in the cytoplasm. There, it gets converted to malate, which can get transported into the mitochondria by a transport protein, mitochondrial 2-oxoglutarate/malate transporter, that is part of yet another cycle shown in the figure above, the asparate arginosuccinate shunt. The introduction of fumarate into the shuttle occurs through the asparate arginosuccinate shunt shown in the figure above. Once malate enters the mitochondrial matrix, it can be directly converted to oxaloacetate.

    Note that these linked cycles require the presence of both mitochondrial and cytoplasmic forms of the several enzyme () and balances between two key amino acids across the mitochondrial divide, gluatamate and asparatate. One such key enzyme is ASpartate Amino Transferase (AST), aka Glutamate Oxalacetate Transaminase (GOT), named for the reverse reaction of aspartate + α−ketoglutarate ↔ oxalacetate + glutamate. As mentioned in the previous chapter section, this key enzyme can be found in the blood if the liver is damaged, so its levels are routinely used in medical test for impaired liver function.

    Another key function of the malate asparate shuttle is bring into the mitochondria reduced "equivalents" of NADH that is produced in the cytoplasm from glycolysis and fatty acid oxidation. There is no membrane transporter for NAD+ or NADH. Instead cytoplasmic malate, produced by the cytosolic reduction of oxalacate by NADH, can be transported into the matrix which it can reform mitochondrial NADH on reconversion to oxalacetate. The NADH can then be used to power ATP production though mitochonrial electron tranpsort/oxidative phosphoyrlation pathways.

    From an energetic perspective, four phosphoanhydrides bonds in 2 ATP molecules are used to produced one urea molecule. This large energy expenditure is partially compensated for by ATP made from the "fumarate equivalents" that enters the mitochondria through the asparate arginosuccinate and asparate arginosuccinate shunts and the passage of NADH through oxidative phosphorylation.