Urease enzyme and its catalytic cycle

bioinorganic chemistry of enzyme activity

1. DEPARTMENT OF CHEMISTRY Presented By GARIMA SHARMA M.Sc. Chemistry 2018MSCH008 SEM - III

2. CONTENT Introduction Structural Properties Characteristics Reaction & Catalytic Cycle Functions Medical Significance and Future Implications UREASE ENZYME : STRUCTURE & FUNCTION

3. 1. Ureases   functionally belong to  superfamily of  amidohydrolases   and phosphotriesterases . 2. Found in numerous bacteria, fungi, algae, plants, and some invertebrates, as well as in soils as soil enzyme. 3. Nickel containing metalloenzyme . 4 . Not synthesized by animals. 5 . Jack bean urease was the first enzyme to be crystallized. 6 . By James. B. Sumner in 1926, Nobel Prize in chemistry in 1946. 7 . Urease catalyzes the hydrolysis of urea to form ammonia and CO2.

4. STRUCTURE Bis- μ - hydroxodimeric nickel

5. The active site of all known 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,magnetic susceptibility experiments have indicated that, in jack bean urease, high spin  octahedrally coordinated Ni(II) ions are weakly  antiferromagnetically  coupled.[ The Ni ions are bridged by a carbamylated lysine and an oxygen donor. The active site contains two nickel ions with an interatomic distance of about 3.5 A. In addition to the bridges, one of the nickel ions (Ni1) is coordinated by two histidine and a water molecule. The coordination of Ni2 is similar to the one of Ni1 and includes two histidine residues, a water molecule and a terminally bound aspartate. STRUCTURAL PROPERTIES

6. In vitro activation also has been achieved with manganese and cobalt in place of nickel .Lead salts are inhibiting . Molecular weight:- 480 manganese and cobalt in place of nickelIn .[12] Lead salts arekDa or 545 kDa for Jack Bean Urease (Hexamer). Optimum pH:- 7.4 Optimum Temperature:- 60 degrees Celsius. Enzymatic Specificity:- Urea and Hydroxyurea . Inhibitors:- Heavy Metals ( Pb - & Pb2+), Fluorine (F). Catalysis hydrolysis of urea to CO2 and NH3.

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

8. Catalytic cycle Urea binds in a bidentate manner with its carbonyl oxygen bound to Ni(1) and one of the amino group bound to Ni(2). Thus replacing two water moieties, leaving only the bridging hydroxide. This hydroxide attacks urea to give the tetrahedral transition state leading to formation of ammonia and carbamate.

9. A MODEL FOR MECHANISM OF UREASE

10. [Ni2(µ-OH)(µ-urea)( bdptz )(urea)(CH3CN)](ClO4)3 [Ni2(µ-OH)(µ-H2O)(bdptz)(µ-OCN)]2(OTs)4 [Ni2(µ-OH)(µ-H2O)( bdptz )(urea)2](ClO4)3 The fact that no dinuclear nickel complex reported thus far has proved capable of hydrolyzing urea to ammonia and carbon dioxide provides an intriguing challenge to the synthetic bioinorganic chemist. The design of a complex that can produce a sufficiently nucleophilic hydroxide ion and at the same time activate a urea molecule by coordination to one or both metal ions should facilitate the successful hydrolysis of urea under the right conditions. So, a dinuclear nickel complex, [Ni2(µ-OH)(µ-H2O)( bdptz )(H2O)2](OTs)3 (where bdptz is the dinucleating ligand 1,4-bis(2,2′-dipyridylmethyl)- phthalazine ), that is capable of hydrolyzing a bound amide substrate by intramolecular attack of a coordinated hydroxide ion. Upon heating, this complex effects the hydrolysis of urea by a pathway involving cyanate ion that is distinct from that previously proposed for the enzyme. These results provide the first direct evidence for a mechanism long considered as an alternative for the enzymatic hydrolysis of urea.

11. Ammonia is the preferred nitrogen source among enteric bacteria and the ureolytic activity of the human gut microbiota hydrolyzes about 15%-30% of the urea synthesized by the human body. (Aoyagi et al.,1966;Walser and Bodenlos,1959) Microbial ureases are an important factor to be considered in dental health. ( Burne et al., 2012). Bacterial ureases in the forestomach of ruminants cleave animal-derived urea allowing the released ammonia to be used as major nitrogen source for the rumen microbiota. ( Laukova and Koniarova , 1995; Pearson and Smith, 1943) Microbial Ureases Function

12. Urea hydrolysis to release ammonia and carbon dioxide is the main physiological role attributed to ureases in plants. (Witte, 2011) Worldwide used as a soil fertilizer, urea can only be assimilated after hydrolysis by urease. ( Sirko and Brodzik,2000) However, the efficiency of urea fertilization can be decreased by higher levels of this soil urease, which can cause massive release of ammonia into the atmosphere, further inducing plant damage by ammonia toxicity and soil pH increase. (Watson et al., 1994) Plant ureases Function

13. People with genetic defects in any enzyme involved in urea formation cannot tolerate protein-rich diets. Amino acids ingested in excess of the minimum daily requirements for protein synthesis are deaminated in the liver, producing free ammonia that cannot be converted to urea and exported into the bloodstream, as ammonia is highly toxic. The absence of urea cycle enzyme can result in hyperammonemia. Although the breakdown of amino acids can have serious health consequences in individuals with urea cycle deficiencies, a protein-free diet is not a treatment option. The absent enzyme activity can often be identified by determining which cycle intermediate is present in especially elevated concentration in the blood and/or urine. A variety of treatments are available for individuals with urea cycle defects. Careful administration of the aromatic acids benzoate or phenyl butyrate in the diet can help lower the level of ammonia in the blood. Medical Significance and Future Implications

14. Hindawi Publishing Corporation, Bioinorganic Chemistry and Applications, Volume 2010, Article ID 364891, 8 pages doi:10.1155/2010/364891 (http://dx.doi.org/10.1155/2010/364891) http://proteopedia.org/wiki/index.php/Urease Plantimals , https://sites.tufts.edu/photosyntheticanimals/urease-mechanism/ Toxicon 110 (2016) 90-109 190–192 (1999) 331–355 (http://users.man.poznan.pl/wojtekr/pdf/wr-lit-22.pdf) J. Am. Chem. Soc. 2000, 122, 9172-9177 References

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