Ureases (EC 3.5.1.5), 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 urease→ 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.
Importance Urease is important because of its role in the nitrogen cycle as a key catalyst in the reaction converting urea to ammonium and CO2. Urease occurs as a soil enzyme, likely because soil microorganisms benefit from the nitrogen made available by urea degradation in the form of ammonium.
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. 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 (αβ)12 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. Other examples of homohexameric structures of plant ureases are those of soybean, pigeon pea and cotton seeds enzymes. 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. The Helicobacter "α" is equivalent to a fusion of the normal bacterial γ-β subunits, while its "β" subunit is equivalent to the normal bacterial α. The three-chain organization is likely ancestral.
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. 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.
Active site The active site of 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. > The Ni(II) pair 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 ligation, including two imidazole ligands per nickel. Urea substrate is proposed to displace aquo ligands. 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. 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 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. The binding of urea to the active site of urease has not been observed.
Proposed mechanisms
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