Urease

Ureases (), functionally, belong to the superfamily of amidohydrolases and phosphotriesterases. Ureases are found in numerous Bacteria, Archaea, fungi, algae, plants, and some invertebrates. Ureases are nickel-containing metalloenzymes of high molecular weight. 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.

These enzymes catalyze the hydrolysis of urea into carbon dioxide and ammonia:

: (NH2)2CO + H2O CO2 + 2NH3

The hydrolysis of urea occurs in two stages. In the first stage, ammonia and carbamic acid are produced. The carbamate spontaneously and rapidly hydrolyzes to ammonia and carbonic acid. Urease activity increases the pH of its environment as ammonia is produced, which is basic.

History

Urease 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.

Structure

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.

The molecular weight is either 480 kDa or 545 kDa for jack-bean urease (calculated mass from the amino acid sequence). 840 amino acids per molecule, of which 90 are cysteine residues.

The optimum pH is 7.4 and optimum temperature is 60 °C. Substrates include urea and hydroxyurea.

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 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. of repeating α-β subunits, each coupled pair of subunits has an active site, for a total of 12 active sites. 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.

Activity

The kcat/Km of urease in the processing of urea is 1014 times greater than the rate of the uncatalyzed elimination reaction of urea. X-ray absorption spectroscopy (XAS) studies of Canavalia ensiformis (jack bean), Klebsiella aerogenes and Sporosarcina pasteurii (formerly known as Bacillus pasteurii) In 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. 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 positively charged group. Blakeley and Zerner proposed this nearby group to be a 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 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.

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. 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. 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.

Infection stones

Infection induced urinary stones are a mixture of struvite (MgNH4PO4•6H2O) and carbonate apatite [Ca10(PO4)6•CO3].

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 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. This was confirmed by decreased ulcer bleeding and ulcer reoccurrence after eradication of the pathogen.

Occurrence and applications in agriculture

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.

Inhibition in fertilizers

The inhibition of urease is a significant goal in agriculture because the rapid breakdown of urea-based fertilizers is wasteful and environmentally damaging. Phenyl phosphorodiamidate and N-(n-butyl)thiophosphoric triamide are two such inhibitors.

Biomineralization

By promoting the formation of calcium carbonate, ureases are potentially useful for biomineralization-inspired processes. Notably, microbiologically induced formation of calcium carbonate can be used in making bioconcrete.

Non-enzymatic action

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 of chemotaxis in bacteria. These activities may be part of a defense mechanism.

Ligands

Inhibitors

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 like H. pylori produce urease as a survival mechanism. Known structural classes of inhibitors include:

Analogues of urea, the strongest being thioureas like 1-(4-chlorophenyl)-3-palmitoylthiourea.
Phosphoramidates, the most commonly used in agriculture (see above).
Hydroquinone and quinones. In medicine, the most interesting are quinolones, already a class of widely used antibiotics.
Some plant metabolites are also urease inhibitors, an example being allicin. These have potential both as environmentally-friendly fertilizer additives and natural drugs.

Extraction

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.