Urease

Ureases, functionally, belong to the superfamily of amidohydrolases and phosphotriestreases. Urease is a nickel-containing enzyme that catalyses the hydrolysis of urea into ammonia and carbon dioxide. More specifically, urease catalyzes the hydrolysis of urea to produce ammonia and carbamate, the carbamate produced is subsequently degraded by spontaneous hydrolysis to produce another ammonia and carbonic acid. Finally with the last degration, the conversion ends with ammonia and carbondioxide as products.

Urease is found in most soils as well as several plants including jack beans and soybeans. There are several different varieties of urease, found in plants, animals and microbes, with different chemical properties and compositions in each type. This enzyme occurs in such different organisms as bacteria, algae, fungi and higher plants. Its primary function is allowing the organism to use urea as a nitrogen source. In plants, urease is involved in systemic nitrogen transport pathways, and is thought to act as a toxic defence protein. In humans, bacterial ureases are important virulence factors in a number of diseases of the urinary tract and gastroduodenal region, including cancer.

It was the first enzyme in history to be purified by James Sumner (1926). Sumner's work was the first demonstration that a pure protein can function as an enzyme, and led eventually to the recognition that most enzymes are in fact proteins, and the award of the Nobel prize in chemistry to Sumner in 1946. The ability of urease to hydrolyze urea into ammonium and carbon dioxide was discovered in 1909 by Takeuchi. Two years later, it was discovered that the enzyme was present in soybeans. In 1913, it was proposed that soybean urease could be applied to quantitatively determine presence of urea. Following the discovery of urease in soybeans, several experiments were performed to show that urease also exists in castor beans. These experiments showed that castor bean urease hydrolyzed less urea than soy bean urease, indicating that castor bean urease is less active than soybean urease. In 1926, Professor James B. Sumner was the first to discover that enzymes were proteins, by isolating and crystallizing urease from jack beans. As a result of his work, urease became the first enzyme discovered. Sumner’s discovery was not accepted for years until another chemist named John H. Northrop was able to isolate an enzyme as well.

The increasing need for specifically removing urea from far different environments has prompted a growing biotechnological interest in this enzyme. Actual or potential applications range from the treatment of industrial wastes or alcoholic beverages to the design of life-support systems for manned space missions. Systems based on immobilised or microencapsulated urease are also being studied for use in haemodialysis.

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 they are cysteine-rich enzymes, resulting in the enzyme molar masses between 190 and 300kDa. All bacterial ureases are solely cytoplasmic, except for Helicobacter pylori urease, which along with its cytoplasmic activity, which has external activity with host cells. In contrast, all plant ureases are cytoplasmic.

The active site of all ureases known are 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. The Nickel ions bridged by a carbamylated lysine through its O-atoms and by a hydroxide ion. Ni is coordinated by N-atoms of histidines residues and one water molecule, is it said to be pseudo square pyramidal. Ni is coordinated by two histidines also through N-atoms and additionally by aspartic acid through its O-atom and 2 water molecules. X-ray absorption spectroscopy (XAS) studies of Canavalia ensiformis (jack bean), Klebsiella aerogenes and Sporosarcina 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 H-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 urease’s, suggesting that all ureases are evolutionary variants of one ancestralenzyme.

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 thecarbonyl carbon making it less of an electrophile making it less reactive to nucleophilic attack.