 3D model of urease fromKlebsiella aerogenes, two Ni2+-ions are shown as green spheres.[1] | |||||||||
| Identifiers | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| EC no. | 3.5.1.5 | ||||||||
| CAS no. | 9002-13-5 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDBPDBePDBsum | ||||||||
| Gene Ontology | AmiGO /QuickGO | ||||||||
| |||||||||
Ureases (EC3.5.1.5), functionally, belong to thesuperfamily ofamidohydrolases and phosphotriesterases.[2] Ureases are found in numerousBacteria,Archaea,fungi,algae, plants, and someinvertebrates. Ureases are nickel-containingmetalloenzymes of high molecular weight.[3] Ureases are important in degrading avian faecal matter, which is rich in uric acid, the breakdown product of which is urea, which is then degraded by urease as described here.
Theseenzymescatalyze thehydrolysis ofurea intocarbon dioxide andammonia:
The hydrolysis ofurea occurs in two stages. In the first stage,ammonia andcarbamic acid are produced. Thecarbamate spontaneously and rapidly hydrolyzes toammonia andcarbonic acid. Urease activity increases thepH of its environment as ammonia is produced, which is basic.
Urease activity was first identified in 1876 byFrédéric Alphonse Musculus as a soluble ferment.[4] In 1926,James B. Sumner, showed that urease is aprotein by examining its crystallized form.[5] Sumner's work was the first demonstration that aprotein can function as anenzyme 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 theNobel prize in chemistry in 1946.[6] The crystal structure of urease was first solved by P. A. Karplus in 1995.[5]
Urease is important because of its role in thenitrogen cycle as a key catalyst in the reaction converting urea to ammonium and CO2. Urease occurs as asoil enzyme, likely because soil microorganisms benefit from the nitrogen made available by urea degradation in the form of ammonium.[7]
A 1984 study focusing on urease fromjack bean found that theactive site contains a pair ofnickel centers.[8]In vitro activation also has been achieved withmanganese andcobalt in place of nickel.[9] Lead salts areinhibiting.
Themolecular weight is either 480kDa or 545kDa for jack-bean urease (calculated mass from the amino acid sequence). 840 amino acids per molecule, of which 90 are cysteine residues.[10]
The optimumpH is 7.4 and optimum temperature is 60 °C. Substrates include urea andhydroxyurea.
Bacterial ureases are composed of three distinct subunits, one large catalytic (α 60–76kDa) and two small (β 8–21 kDa, γ 6–14 kDa) commonly forming (αβγ)3 trimersstoichiometry 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.[10]
An exceptional urease is obtained fromHelicobacter sp.. These are composed of two subunits, α(26–31 kDa)-β(61–66 kDa). These subunits form a supramolecular (αβ)12dodecameric complex.[11] of repeating α-β subunits, each coupled pair of subunits has an active site, for a total of 12 active sites.[11] It plays an essential function for survival, neutralizinggastric acid by allowingurea to enter intoperiplasm via aproton-gated urea channel.[12] The presence of urease is used in the diagnosis ofHelicobacter species.
All bacterial ureases are solely cytoplasmic, except for those inHelicobacter pylori, which along with its cytoplasmic activity, has external activity with host cells. In contrast, all plant ureases are cytoplasmic.[10]
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 thehexamer with the 12 nickel ions is 545.34 kDa. Other examples of homohexameric structures of plant ureases are those of soybean, pigeon pea and cotton seeds enzymes.[10]
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 single plant urease chain is equivalent to a fused γ-β-α organization. TheHelicobacter "α" is equivalent to a fusion of the normal bacterial γ-β subunits, while its "β" subunit is equivalent to the normal bacterial α.[10] The three-chain organization is likely ancestral.[13]
Thekcat/Km of urease in the processing ofurea is 1014 times greater than the rate of the uncatalyzed elimination reaction ofurea.[5] There are many reasons for this observation in nature. The proximity ofurea to active groups in the active site along with the correct orientation of urea allowhydrolysis 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 itsresonance energy, which has been estimated at 30–40 kcal/mol.[5] This is because thezwitterionic resonance forms all donate electrons to thecarbonyl carbon making it less of anelectrophile making it less reactive to nucleophilic attack.[5]
Theactive site of ureases is located in the α (alpha)subunits. It is a bis-μ-hydroxo dimericnickel center, with an interatomic distance of ~3.5 Å.[5] > The Ni(II) pair are weaklyantiferromagnetically coupled.[14]X-ray absorption spectroscopy (XAS) studies ofCanavalia ensiformis (jack bean),Klebsiella aerogenes andSporosarcina pasteurii (formerly known asBacillus pasteurii)[15] confirm 5–6 coordinate nickel ions with exclusively O/N ligation, including twoimidazole ligands per nickel.[9] Urea substrate is proposed to displaceaquo ligands.
Water molecules located towards the opening of the active site form a tetrahedral cluster that fills the cavity site throughhydrogen bonds. Some amino acid residues are proposed to form mobile flap of the site, which gate for the substrate.[3] 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.[16] InSporosarcina pasteurii urease, the flap was found in the open conformation, while its closed conformation is apparently needed for the reaction.[15]
When compared, the α subunits ofHelicobacter pylori urease and other bacterial ureases align with the jack bean ureases.[16]
The binding of urea to the active site of urease has not been observed.[10]
One mechanism for the catalysis of this reaction by urease was proposed by Blakely and Zerner.[17] It begins with a nucleophilic attack by thecarbonyl oxygen of theurea 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 theUrea molecule creates a double bond with the central carbon, and the resulting NH2− of the coordinated substrate interacts with a nearby positively charged group. Blakeley and Zerner proposed this nearby group to be aCarboxylate ion, although deprotonated carboxylates are negatively charged.
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 acysteine located near the active site. A hydrogen bonds to one of the nitrogen atoms, breaking its bond with carbon, and releasing anNH3 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.
Thecarbamate produced then spontaneously degrades to produce another ammonia andcarbonic acid.[18]
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.[5] From the crystal structures fromK. 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.[19] 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.[5] 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.[5] 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.[5] 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.[5]
The mechanism proposed by Ciurli and Mangani[20] 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 twonickel ions in the active site.[15] One of which binds and activates urea, the other nickel ion binds and activates the nucleophilic water molecule.[15] 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 ahydrogen-bonding network, orienting the substrate into the catalytic cavity.[15] Urea binds to the five-coordinated nickel (Ni1) with the carbonyloxygen atom. It approaches the six-coordinated nickel (Ni2) with one of its amino groups and subsequently bridges the two nickel centers.[15] 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.[15] 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.[15] Urea is a very poorchelating ligand due to lowLewis 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.[15] 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.[15] The main structural difference between the Ciurli/Mangani mechanism and the other two is that it incorporates anitrogen, oxygen bridging urea that is attacked by a bridginghydroxide.[18]
Bacterial ureases are often the mode ofpathogenesis for many medical conditions. They are associated withhepatic encephalopathy /Hepatic coma, infection stones, and peptic ulceration.[21]
Infection induced urinary stones are a mixture ofstruvite (MgNH4PO4•6H2O) andcarbonateapatite [Ca10(PO4)6•CO3].[21] These polyvalent ions are soluble but become insoluble whenammonia is produced from microbial urease duringureahydrolysis, as this increases the surrounding environmentspH from roughly 6.5 to 9.[21] The resultant alkalinization results in stonecrystallization.[21] In humans the microbial urease,Proteus mirabilis, is the most common in infection induced urinary stones.[22]
Studies have shown thatHelicobacter pylori along withcirrhosis of the liver causehepatic encephalopathy andhepatic coma.[23]Helicobacter pylori release microbial ureases into the stomach. The urease hydrolyzesurea to produceammonia andcarbonic acid. As the bacteria are localized to the stomachammonia produced is readily taken up by thecirculatory system from the gastriclumen.[23] This results in elevatedammonia levels in the blood, a condition known ashyperammonemia; eradication ofHelicobacter pylori show marked decreases inammonia levels.[23]
Helicobacter pylori is also the cause of peptic ulcers with its manifestation in 55–68% reported cases.[24] This was confirmed by decreasedulcer bleeding andulcer reoccurrence after eradication of thepathogen.[24] In the stomach there is an increase inpH of the mucosal lining as a result ofureahydrolysis, which prevents movement ofhydrogen ions between gastric glands and gastriclumen.[21] In addition, the highammonia concentrations have an effect on intercellulartight junctions increasing permeability and also disrupting the gastricmucous membrane of the stomach.[21][25]
Urea is found naturally in the environment and is also artificially introduced, comprising more than half of all synthetic nitrogen fertilizers used globally.[26] Heavy use of urea is thought to promoteeutrophication, despite the observation that urea is rapidly transformed by microbial ureases, and thus usually does not persist.[27] 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.[28]
The inhibition of urease is a significant goal in agriculture because the rapid breakdown of urea-based fertilizers is wasteful and environmentally damaging.[29]Phenyl phosphorodiamidate andN-(n-butyl)thiophosphoric triamide are two such inhibitors.[30]
By promoting the formation ofcalcium carbonate, ureases are potentially useful forbiomineralization-inspired processes.[31] Notably, microbiologically induced formation of calcium carbonate can be used in makingbioconcrete.[32]
In addition to acting as an enzyme, some ureases (especially plant ones) have additional effects that persist even when the catalytic function is disabled. These include entomotoxicity, inhibition of fungi,neurotoxicity in mammals, promotion of endocytosis and inflammatory eicosanoid production in mammals, and induction ofchemotaxis in bacteria. These activities may be part of a defense mechanism.[13]
Urease insect-toxicity was originally noted in canatoxin, an orthologous isoform of jack bean urease. Digestion of the peptide identified a 10-kDa portion most responsible for this effect, termed jaburetox. An analogous portion from the soybean urease is named soyuretox. Studies on insects show that the entire protein is toxic without needing any digestion, however. Nevertheless, the "uretox" peptides, being more concentrated in toxicity, show promise asbiopesticides.[13]
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:
A wide range of urease inhibitors of different structural families are known. Inhibition of urease is not only of interest to agriculture, but also to medicine as pathogens likeH. pylori produce urease as a survival mechanism. Known structural classes of inhibitors include:[34][35]
 | This articleis missing information about applications of urease. Please expand the article to include this information. Further details may exist on thetalk page.(May 2022) |
First isolated as a crystal in 1926 by Sumner, using acetone solvation and centrifuging.[37] Modern biochemistry has increased its demand for urease.Jack bean meal,[38]watermelon seeds,[39] andpea seeds[40] have all proven useful sources of urease.
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