Editing Urease (section)

==Activity==

The ''k''<sub>cat</sub>/''K''<sub>m</sub> of urease in the processing of [[urea]] is 10<sup>14</sup> times greater than the rate of the uncatalyzed elimination reaction of [[urea]].<ref name="karplus"/> 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.<ref name="karplus"/> 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.<ref name="karplus"/>

===Active site===

The [[active site]] of ureases is located in the α (alpha) [[Protein subunit|subunits]]. It is a bis-μ-hydroxo dimeric [[nickel]] center, with an interatomic distance of ~3.5 Å.<ref name="karplus" /> > The Ni(II) pair are weakly [[Antiferromagnetism|antiferromagnetically]] coupled.<ref>{{cite journal | vauthors = Ciurli S, Benini S, Rypniewski WR, Wilson KS, Miletti S, Mangani S | title = Structural properties of the nickel ions in urease: novel insights into the catalytic and inhibition mechanisms | journal = Coordination Chemistry Reviews | year = 1999 | volume = 190–192 | pages = 331–355 | doi = 10.1016/S0010-8545(99)00093-4 }}</ref> [[X-ray absorption spectroscopy]] (XAS) studies of ''[[Canavalia ensiformis]]''  (jack bean), ''Klebsiella aerogenes''  and ''[[Sporosarcina pasteurii]]'' (formerly known as ''Bacillus pasteurii'')<ref name="Benini, S. 1999"/> confirm 5–6 coordinate nickel ions with exclusively O/N ligation, including two [[imidazole]] ligands per nickel.<ref name="pmid20046957" /> Urea substrate is proposed to displace [[aquo ligand]]s.

Water molecules located towards the opening of the active site form a tetrahedral cluster that fills the cavity site through [[hydrogen bonds]].  Some amino acid residues are proposed to form mobile flap of the site, which gate for the substrate.<ref name="Krajewska" /> 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.<ref name="Martin">{{cite journal | vauthors = Martin PR, Hausinger RP | title = Site-directed mutagenesis of the active site cysteine in ''Klebsiella aerogenes'' urease | journal = The Journal of Biological Chemistry | volume = 267 | issue = 28 | pages = 20024–7 | date = Oct 5, 1992 | doi = 10.1016/S0021-9258(19)88659-3 | pmid = 1400317 | doi-access = free }}</ref> In ''[[Sporosarcina pasteurii]]'' urease, the flap was found in the open conformation, while its closed conformation is apparently needed for the reaction.<ref name="Benini, S. 1999">{{cite journal | vauthors = Benini S, Rypniewski WR, Wilson KS, Miletti S, Ciurli S, Mangani S | title = A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels | journal = Structure | volume = 7 | issue = 2 | pages = 205–216 | date = 31 January 1999 | pmid = 10368287 | doi = 10.1016/S0969-2126(99)80026-4 | doi-access = free }}</ref>

When compared, the α subunits of ''[[Helicobacter pylori]]'' urease and other bacterial ureases align with the jack bean ureases.<ref name="Martin" />

The binding of urea to the active site of urease has not been observed.<ref name="Molecular Catalysis B 2009"/>

===Proposed mechanisms===

====Blakeley/Zerner====

One mechanism for the catalysis of this reaction by urease was proposed by Blakely and Zerner.<ref name="pmid6788353">{{cite journal | vauthors = Dixon NE, Riddles PW, Gazzola C, Blakeley RL, Zerner B | title = Jack Jack Bean Urease (EC3.5.1.5). V. On the Mechanism of action of urease on urea, formamide, acetamide,N-methylurea, and related compounds | journal = Canadian Journal of Biochemistry | volume = 58 | issue = 12 | pages = 1335–1344 | year = 1979 | pmid = 6788353 | doi = 10.1139/o80-181 }}</ref> 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 NH<sub>2</sub><sup>−</sup> of the coordinated substrate interacts with a nearby positively charged group. Blakeley and Zerner proposed this nearby group to be a [[Carboxylate|Carboxylate 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 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 [[carbamate]] produced then spontaneously degrades to produce another ammonia and [[carbonic acid]].<ref name="Zimmer">{{cite journal | vauthors = Zimmer M | title = Molecular mechanics evaluation of the proposed mechanisms for the degradation of urea by urease | journal = J Biomol Struct Dyn | volume = 17 | issue = 5 | pages = 787–97 | date = Apr 2000 | pmid = 10798524 | doi = 10.1080/07391102.2000.10506568 | s2cid = 41497756 }}</ref>

====Hausinger/Karplus====

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.<ref name="karplus"/>  From the crystal structures from ''K. aerogenes'' urease, it was argued that the general base used in the Blakely mechanism, His<sup>320</sup>, 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.<ref name="Jabri">{{cite journal | vauthors = Jabri E, Carr MB, Hausinger RP, Karplus PA | title = The crystal structure of urease from Klebsiella aerogenes | journal = Science | volume = 268 | issue = 5213 | pages = 998–1004 | date = May 19, 1995 | pmid = 7754395 | doi = 10.1126/science.7754395 | bibcode = 1995Sci...268..998J }}</ref> Hausinger and Karplus suggests a reverse protonation scheme, where a protonated form of the His<sup>320</sup> ligand plays the role of the general acid and the Ni2-bound water is already in the deprotonated state.<ref name="karplus"/>  The mechanism follows the same path, with the general base omitted (as there is no more need for it) and His<sup>320</sup> donating its proton to form the ammonia molecule, which is then released from the enzyme.  While the majority of the His<sup>320</sup> 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.<ref name="karplus"/>  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.<ref name="karplus"/>  Placing the His<sup>320</sup> 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.<ref name="karplus"/>

====Ciurli/Mangani====

The mechanism proposed by Ciurli and Mangani<ref name="pmid21542631">{{cite journal | vauthors = Zambelli B, Musiani F, Benini S, Ciurli S | title = Chemistry of Ni2+ in Urease: Sensing, Trafficking, and Catalysis | journal = Accounts of Chemical Research | volume = 44 | issue = 7 | pages = 520–530 | date = 19 July 2011 | pmid = 21542631 | doi = 10.1021/ar200041k }}</ref> 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.<ref name="Benini, S. 1999" /> One of which binds and activates urea, the other nickel ion binds and activates the nucleophilic water molecule.<ref name="Benini, S. 1999"/>  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.<ref name="Benini, S. 1999"/>  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.<ref name="Benini, S. 1999"/>  The binding of the urea carbonyl oxygen atom to Ni1 is stabilized through the protonation state of His<sup>α222</sup> Nԑ. Additionally, the conformational change from the open to closed state of the mobile flap generates a rearrangement of Ala<sup>α222</sup> carbonyl group in such a way that its oxygen atom points to Ni2.<ref name="Benini, S. 1999"/>  The Ala<sup>α170</sup> and Ala<sup>α366</sup> are now oriented in a way that their carbonyl groups act as hydrogen-bond acceptors towards NH<sub>2</sub> group of urea, thus aiding its binding to Ni2.<ref name="Benini, S. 1999"/>  Urea is a very poor [[chelating ligand]] due to low [[Lewis base]] character of its NH<sub>2</sub> groups. However the carbonyl oxygens of Ala<sup>α170</sup> and Ala<sup>α366</sup> enhance the basicity of the NH<sub>2</sub> groups and allow for binding to Ni2.<ref name="Benini, S. 1999"/> 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.<ref name="Benini, S. 1999"/>  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]].<ref name=Zimmer />

===Action in pathogenesis===

Bacterial ureases are often the mode of [[pathogenesis]] for many medical conditions. They are associated with [[hepatic encephalopathy]] / [[Hepatic coma]], infection stones, and peptic ulceration.<ref name="mobley">{{cite journal | vauthors = Mobley HL, Hausinger RP | title = Microbial ureases: significance, regulation, and molecular characterization | journal = Microbiological Reviews | volume = 53 | issue = 1 | pages = 85–108 | date = March 1989 | doi = 10.1128/MMBR.53.1.85-108.1989 | pmid = 2651866 | pmc = 372718 }}</ref>

====Infection stones====

Infection induced urinary stones are a mixture of [[struvite]] (MgNH<sub>4</sub>PO<sub>4</sub>•6H<sub>2</sub>O) and [[carbonate]] [[apatite]] [Ca<sub>10</sub>(PO<sub>4</sub>)6•CO<sub>3</sub>].<ref name="mobley"/> 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.<ref name="mobley"/> The resultant alkalinization results in stone [[crystallization]].<ref name="mobley"/> In humans the microbial urease, ''Proteus mirabilis'', is the most common in infection induced urinary stones.<ref name="pmid3524996">{{cite journal | vauthors = Rosenstein IJ | title = Urinary Calculi: Microbiological and Crystallographic Studies | journal = Critical Reviews in Clinical Laboratory Sciences | volume = 23 | issue = 3 | pages = 245–277 | date = 1 January 1986 | pmid = 3524996 | doi = 10.3109/10408368609165802 }}</ref>

====Urease in hepatic encephalopathy / hepatic coma====

Studies have shown that ''[[Helicobacter pylori]]'' along with [[cirrhosis]] of the liver cause [[hepatic encephalopathy]] and [[hepatic coma]].<ref name="agrawal">{{cite journal | vauthors = Agrawal A, Gupta A, Chandra M, Koowar S | title = Role of Helicobacter pylori infection in the pathogenesis of minimal hepatic encephalopathy and effect of its eradication | journal = Indian Journal of Gastroenterology | volume = 30 | issue = 1 | pages = 29–32 | date = 17 March 2011 | pmid = 21416318 | doi = 10.1007/s12664-011-0087-7 | s2cid = 25452909 }}</ref> ''Helicobacter pylori''  release microbial ureases into the stomach. The urease hydrolyzes [[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 (anatomy)|lumen]].<ref name="agrawal"/> This results in elevated [[ammonia]] levels in the blood, a condition known as [[hyperammonemia]]; eradication of ''Helicobacter pylori'' show marked decreases in [[ammonia]] levels.<ref name="agrawal"/>

====Urease in peptic ulcers====

''Helicobacter pylori'' is also the cause of peptic ulcers with its manifestation in 55–68% reported cases.<ref name="tang">{{cite journal | vauthors = Tang JH, Liu NJ, Cheng HT, Lee CS, Chu YY, Sung KF, Lin CH, Tsou YK, Lien JM, Cheng CL | title = Endoscopic diagnosis of Helicobacter pylori infection by rapid urease test in bleeding peptic ulcers: a prospective case-control study | journal = Journal of Clinical Gastroenterology | volume = 43 | issue = 2 | pages = 133–9 | date = February 2009 | pmid = 19230239 | doi = 10.1097/MCG.0b013e31816466ec | s2cid = 27784917 }}</ref>  This was confirmed by decreased [[ulcer]] bleeding and [[ulcer]] reoccurrence after eradication of the [[pathogen]].<ref name="tang"/> 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 (anatomy)|lumen]].<ref name="mobley"/>  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.<ref name="mobley"/><ref>{{cite journal | vauthors = Caron TJ, Scott KE, Fox JG, Hagen SJ | title = Tight junction disruption: Helicobacter pylori and dysregulation of the gastric mucosal barrier | journal = World Journal of Gastroenterology | volume = 21 | issue = 40 | pages = 11411–27 | date = October 2015 | pmid = 26523106 | pmc = 4616217 | doi = 10.3748/wjg.v21.i40.11411 | doi-access = free }}</ref>