Ureases (EC 184.108.40.206), functionally, belong to the superfamily of amidohydrolases and phosphotriesterases. Ureases are found in numerous bacteria, fungi, algae, plants, and some invertebrates, as well as in soils, as a soil enzyme. They are nickel-containing metalloenzymes of high molecular weight.
3D model of urease from Klebsiella aerogenes, two Ni2+-ions are shown as green spheres. 
|PDB structures||RCSB PDB PDBe PDBsum|
|Gene Ontology||AmiGO / QuickGO|
- (NH2)2CO + H2O → CO2 + 2NH3
The hydrolysis of urea occurs in two stages. In the first stage, ammonia and carbamate are produced. The carbamate spontaneously and rapidly hydrolyzes to ammonia and carbonic acid. Urease activity increase the pH of its environment as it produces ammonia, which is basic.
Urease is also found in mammals and humans which is considered to be very harmful to the mammals due to production of the toxic ammonia product in the mammalian cells. However, mammalian cells does not produce urease in fact, the source is the various bacterias in the body, specifically the intestine. European hare (Lepus europaeus),a class of Mammalia, was discovered to have high urease activity in their large intestine, a part of gastrointestinal tract. Previously, other mammals i.e. rats, pigs and rabbits, with postgastric fermentation were detected with lower urease activity compared to European Hare. In human kidneys, urea is present in order for everyday functions and is estimated that per day, a healthy adult excretes about 10 to 30 g of urea. Other than urea being found in urine, it is also present in sweat, blood serum and stomach. Inside the mitochondria of a liver cell, excess ammonia is converted to urea through the urea cycle and if some excess ammonia is still present in the mitochondria, then it gets used up for protein synthesis. There are specific tissues involved during urea processing which are epithelial, extrahepatic and muscle tissues. With the production of ammonia and amino acids, the cell proteins are broken down by proteolytic enzymes already present in the muscle tissue. Similarly, identical cell proteins are predicted to convert previously broken down ammonia into urea. Once the urea is formed in the liver, it is excreted through urine after passing from bloodstream and the kidneys.
Its activity was first identified in 1876 by Frédéric Alphonse Musculus as a soluble ferment. In 1926, James B. Sumner, showed that urease is a protein by examining its crystallized form. Sumner's work was the first demonstration that a protein can function as an enzyme and led eventually to the recognition that most enzymes are in fact proteins. Urease was the first enzyme crystallized. For this work, Sumner was awarded the Nobel prize in chemistry in 1946. The crystal structure of urease was first solved by P. A. Karplus in 1995.
A 1984 study focusing on urease from jack bean found that the active site contains a pair of nickel centers. In vitro activation also has been achieved with manganese and cobalt in place of nickel. Lead salts are inhibiting.
Bacterial ureases are composed of three distinct subunits, one large (α 60–76kDa) and two small (β 8–21 kDa, γ 6–14 kDa) commonly forming (αβγ)3 trimers stoichiometry with a 2-fold symmetric structure (note that the image above gives the structure of the asymmetric unit, one-third of the true biological assembly), they are cysteine-rich enzymes, resulting in the enzyme molar masses between 190 and 300kDa.
An exceptional urease is obtained from Helicobacter sp.. These are composed of two subunits, α(26–31 kDa)-β(61–66 kDa). These subunits form a supramolecular dodecameric complex. of repeating α-β subunits, each coupled pair of subunits has an active site, for a total of 12 active sites. ( ). It plays an essential function for survival, neutralizing gastric acid by allowing urea to enter into periplasm via a proton-gated urea channel. The presence of urease is used in the diagnosis of Helicobacter species.
All bacterial ureases are solely cytoplasmic, except for those in Helicobacter pylori, which along with its cytoplasmic activity, has external activity with host cells. In contrast, all plant ureases are cytoplasmic.
Fungal and plant ureases are made up of identical subunits (~90 kDa each), most commonly assembled as trimers and hexamers. For example, jack bean urease has two structural and one catalytic subunit. The α subunit contains the active site, it is composed of 840 amino acids per molecule (90 cysteines), its molecular mass without Ni(II) ions amounting to 90.77 kDa. The mass of the hexamer with the 12 nickel ions is 545.34 kDa. It is structurally related to the (αβγ)3 trimer of bacterial ureases. Other examples of homohexameric structures of plant ureases are those of soybean, pigeon pea and cotton seeds enzymes.
It is important to note, that although composed of different types of subunits, ureases from different sources extending from bacteria to plants and fungi exhibit high homology of amino acid sequences.
The kcat/Km of urease in the processing of urea is 1014 times greater than the rate of the uncatalyzed elimination reaction of urea. There are many reasons for this observation in nature. The proximity of urea to active groups in the active site along with the correct orientation of urea allow hydrolysis to occur rapidly. Urea alone is very stable due to the resonance forms it can adopt. The stability of urea is understood to be due to its resonance energy, which has been estimated at 30–40 kcal/mol. This is because the zwitterionic resonance forms all donate electrons to the carbonyl carbon making it less of an electrophile making it less reactive to nucleophilic attack.
The active site of all known ureases is located in the α (alpha) subunits. It is a bis-μ-hydroxo dimeric nickel center, with an interatomic distance of ~3.5 Å, magnetic susceptibility experiments have indicated that, in jack bean urease, high spin octahedrally coordinated Ni(II) ions are weakly antiferromagnetically coupled. X-ray absorption spectroscopy (XAS) studies of Canavalia ensiformis (jack bean), Klebsiella aerogenes and Sporosarcina pasteurii (formerly known as Bacillus pasteurii) confirm 5–6 coordinate nickel ions with exclusively O/N ligands (two imidazoles per nickel).
The water molecules are located towards the opening of the active site and form a tetrahedral cluster that fills the cavity site through hydrogen bonds, and it's here where urea binds to the active site for the reaction, displacing the water molecules. The amino acid residues participate in the substrate binding, mainly through hydrogen bonding, stabilize the catalytic transition state and accelerate the reaction. Additionally, the amino acid residues involved in the architecture of the active site compose part of the mobile flap of the site, which is said to act as a gate for the substrate. Cysteine residues are common in the flap region of the enzymes, which have been determined not to be essential in catalysis, although involved in positioning other key residues in the active site appropriately. In the structure of Sporosarcina pasteurii urease the flap was found in the open conformation, while its closed conformation is apparently needed for the reaction.
When compared, the α subunits of Helicobacter pylori urease and other bacterial ureases align with the jack bean ureases, suggesting that all ureases are evolutionary variants of one ancestral enzyme.
It is important to note that the coordination of urea to the active site of urease has never been observed in a resting state of the enzyme.
One mechanism for the catalysis of this reaction by urease was proposed by Blakely and Zerner. It begins with a nucleophilic attack by the carbonyl oxygen of the urea molecule onto the 5-coordinate Ni (Ni-1). A weakly coordinated water ligand is displaced in its place. A lone pair of electrons from one of the nitrogen atoms on the Urea molecule creates a double bond with the central carbon, and the resulting NH2+ of the coordinated substrate interacts with a nearby negatively charged group. Blakeley and Zerner proposed this nearby group to be a Carboxylate ion
A hydroxide ligand on the six coordinate Ni is deprotonated by a base. The carbonyl carbon is subsequently attacked by the electronegative oxygen. A pair of electrons from the nitrogen-carbon double bond returns to the nitrogen and neutralizes the charge on it, while the now 4-coordinate carbon assumes an intermediate tetrahedral orientation.
The breakdown of this intermediate is then helped by a sulfhydryl group of a cysteine located near the active site. A hydrogen bonds to one of the nitrogen atoms, breaking its bond with carbon, and releasing an NH3 molecule. Simultaneously, the bond between the oxygen and the 6-coordinate nickel is broken. This leaves a carbamate ion coordinated to the 5-coordinate Ni, which is then displaced by a water molecule, regenerating the enzyme.
The mechanism proposed by Hausinger and Karplus attempts to revise some of the issues apparent in the Blakely and Zerner pathway, and focuses on the positions of the side chains making up the urea-binding pocket. From the crystal structures from K. aerogenes urease, it was argued that the general base used in the Blakely mechanism, His320, was too far away from the Ni2-bound water to deprotonate in order to form the attacking hydroxide moiety. In addition, the general acidic ligand required to protonate the urea nitrogen was not identified. Hausinger and Karplus suggests a reverse protonation scheme, where a protonated form of the His320 ligand plays the role of the general acid and the Ni2-bound water is already in the deprotonated state. The mechanism follows the same path, with the general base omitted (as there is no more need for it) and His320 donating its proton to form the ammonia molecule, which is then released from the enzyme. While the majority of the His320 ligands and bound water will not be in their active forms (protonated and deprotonated, respectively,) it was calculated that approximately 0.3% of total urease enzyme would be active at any one time. While logically, this would imply that the enzyme is not very efficient, contrary to established knowledge, usage of the reverse protonation scheme provides an advantage in increased reactivity for the active form, balancing out the disadvantage. Placing the His320 ligand as an essential component in the mechanism also takes into account the mobile flap region of the enzyme. As this histidine ligand is part of the mobile flap, binding of the urea substrate for catalysis closes this flap over the active site and with the addition of the hydrogen bonding pattern to urea from other ligands in the pocket, speaks to the selectivity of the urease enzyme for urea.
The mechanism proposed by Ciurli and Mangani is one of the more recent and currently accepted views of the mechanism of urease and is based primarily on the different roles of the two nickel ions in the active site. One of which binds and activates urea, the other nickel ion binds and activates the nucleophilic water molecule. With regards to this proposal, urea enters the active site cavity when the mobile ‘flap’ (which allows for the entrance of urea into the active site) is open. Stability of the binding of urea to the active site is achieved via a hydrogen-bonding network, orienting the substrate into the catalytic cavity. Urea binds to the five-coordinated nickel (Ni1) with the carbonyl oxygen atom. It approaches the six-coordinated nickel (Ni2) with one of its amino groups and subsequently bridges the two nickel centers. The binding of the urea carbonyl oxygen atom to Ni1 is stabilized through the protonation state of Hisα222 Nԑ. Additionally, the conformational change from the open to closed state of the mobile flap generates a rearrangement of Alaα222 carbonyl group in such a way that its oxygen atom points to Ni2. The Alaα170 and Alaα366 are now oriented in a way that their carbonyl groups act as hydrogen-bond acceptors towards NH2 group of urea, thus aiding its binding to Ni2. Urea is a very poor chelating ligand due to low Lewis base character of its NH2 groups. However the carbonyl oxygens of Alaα170 and Alaα366 enhance the basicity of the NH2 groups and allow for binding to Ni2. Therefore, in this proposed mechanism, the positioning of urea in the active site is induced by the structural features of the active site residues which are positioned to act as hydrogen-bond donors in the vicinity of Ni1 and as acceptors in the vicinity of Ni2. The main structural difference between the Ciurli/Mangani mechanism and the other two is that it incorporates a nitrogen, oxygen bridging urea that is attacked by a bridging hydroxide.
Action in pathogenesisEdit
Infection induced urinary stones are a mixture of struvite (MgNH4PO4•6H2O) and carbonate apatite [Ca10(PO4)6•CO3]. These polyvalent ions are soluble but become insoluble when ammonia is produced from microbial urease during urea hydrolysis, as this increases the surrounding environments pH from roughly 6.5 to 9. The resultant alkalinization results in stone crystallization. In humans the microbial urease, Proteus mirabilis, is the most common in infection induced urinary stones.
Urease in hepatic encephalopathy / hepatic coma
Studies have shown that Helicobacter pylori along with cirrhosis of the liver cause hepatic encephalopathy and hepatic coma. Helicobacter pylori are microbial ureases found in the stomach. As ureases they hydrolyze urea to produce ammonia and carbonic acid. As the bacteria are localized to the stomach ammonia produced is readily taken up by the circulatory system from the gastric lumen. This results in elevated ammonia levels in the blood and is coined as hyperammonemia, eradication of Heliobacter pylori show marked decreases in ammonia levels.
Urease in peptic ulcers
Helicobacter pylori is also the cause of peptic ulcers with its manifestation in 55–68% reported cases. This was confirmed by decreased ulcer bleeding and ulcer reoccurrence after eradication of the pathogen. In the stomach there is an increase in pH of the mucosal lining as a result of urea hydrolysis, which prevents movement of hydrogen ions between gastric glands and gastric lumen. In addition, the high ammonia concentrations have an effect on intercellular tight junctions increasing permeability and also disrupting the gastric mucous membrane of the stomach.
Occurrence and potential applicationsEdit
Urea is found naturally in the environment and is also artificially introduced, comprising more than half of all synthetic nitrogen fertilizers used globally. Heavy use of urea is thought to promote eutrophication, despite the observation that urea is rapidly transformed by microbial ureases, and thus usually does not persist. Environmental urease activity is often measured as an indicator of the health of microbial communities. In the absence of plants, urease activity in soil is generally attributed to heterotrophic microorganisms, although it has been demonstrated that some chemoautotrophic ammonium oxidizing bacteria are capable of growth on urea as a sole source of carbon, nitrogen, and energy.
By promoting the formation of calcium carbonate, ureases are potentially useful for biomineralization-inspired processes. Notably, micro-biologically induced formation of calcium carbonate can be used in making bioconcrete.
As diagnostic testEdit
Many gastrointestinal or urinary tract pathogens produce urease, enabling the detection of urease to be used as a diagnostic to detect presence of pathogens.
Urease-positive pathogens include:
- Proteus mirabilis and Proteus vulgaris
- Ureaplasma urealyticum, a relative of Mycoplasma spp.
- Corynebacterium urealyticum
- Cryptococcus spp., an opportunistic fungus
- Helicobacter pylori
- Certain Enteric bacteria including Proteus spp., Klebsiella spp., Morganella, Providencia, and possibly Serratia spp.
- Staphylococcus saprophyticus
- Staphylococcus aureus
First isolated as a crystal in 1926 by Sumner, using acetone solvation and centrifuging. Modern biochemistry has increased its demand for urease. Jack bean meal, watermelon seeds, and pea seeds have all proven useful sources of urease.
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