basic of Urease mimic

1. Urease Mimics
By
Research Scholar
M. Sathiyendran

2. Contents
Introduction
• Enzyme
• Urease structure
• Urease function
Article
• Abstract
• Procedures
• Spectral studies
• Conclusion

3. What is enzyme?
 Macromolecular biological catalysts
 It increase the rate of a reaction by lower activation energy
 All enzymes are proteins, but not all proteins are enzymes
 Used as a laundry detergents in industrial processes
 It transform starch into sugar without any side effects
 It work at low temperature and moderate PH and more stable
 biodegradable and environmentally friendly
 enzymes are made of up to a million amino acids, Each amino acid is bonded to the next by
chemical bonds
 inside inhibitors = drug and poisons, outside inhibitors = temperature and PH

4. Introduction of Urease
 It’s a Nickel-dependent metalloenzyme and was first crystallized
 Urease substrate (Urea) was the First synthesized organic compound
 urea is short-lived and rapidly metabolized by microbial activities
Urease is synthesized by plants, some bacteria, and fungi
 It catalyzes simple hydrolysis of urea
 The increased ammonia level has the great implications in agriculture and medical field
 and it play a vital role in pathogenic microorganisms, urinary stone formation, environmental
cycling of various nitrogen compounds, seed germination by degrading urea
 One of the most common bacterial urease is the Helicobacter pylori since it has been implicated in
peptic ulcers and stomach cancer
Molecular weight: 480 kDa or 545 kDa, Optimum PH : 7.4
Optimum Temperature: 600C, Inhibitors: Heavy Metals (Pb- & Pb2+)

5. Structure of Urease
X-ray crystallography of the enzyme active site

6.  The urease active site has two Ni atoms bridged by oxygen atoms a carboxylated lysine and a
hydroxyl group.
 Each nickel coordinates two histidine residues and a water molecule
The coordination sphere of Ni(2) is completed by an additionally terminated aspartate
 Penta-coordinated Ni (1) containing distorted square-pyramidal geometry, another one Ni (2) ion
has hexa-coordinated with a distorted octahedral geometry
 It has been confirmed that carbon dioxide is required for nickel binding to apo-urease and lysine
residue reacts with carbon dioxide and converts to a carbamate which captures the nickel ions into
the active site.
 One metal site binds the urea carbonyl oxygen and enhances the electrophilicity of the carbon
atom,
 The second metal increases the nucleophilicity of a water molecule
Protein side chains serve additional acid/base roles in urea hydrolysis
 Ni-Ni distance of ~3.26 Å, weak antiferromagnetic exchange coupling between the nickel atoms
 An additional water molecule is part of a hydrogen-bonding network completing a tetrahedral
cluster of four water/hydroxide molecules
Structural Explanation

7. About Ni2+
 The major use of nickel is making alloys, stainless steel, rechargeable batteries, catalysts, coinage
foundry products, and plating.
these are the good conductor of heat and electricity but resistance against corrosion
 tetrahedral & diamagnetic
 Surgical implants, medical tools, health care equipment and fixtures, as well as dental tools and
implants and Anemia
An uptake of too large quantities of nickel has the following consequences:
 lung cancer, nose cancer, larynx cancer and prostate cancer
Sickness and dizziness after exposure to nickel gas
Lung embolism, Respiratory failure
Birth defects, Asthma
Allergic reactions such as skin rashes, mainly from jewellery
Heart disorders

8. Role of nickel in urease

9.  The hydroxyl group bound to Ni-2 attacks urea
 The carbonyl group is polarized by coordination to Ni1, forming a tetrahedral
intermediate
 That releases ammonia with His 320 acting as a general acid.
 The bridging hydroxyl group attacks urea, bound with its carbonyl group
coordinated to Ni1 and an amine interacting with Ni2, and the hydroxyl proton
transfers to the released ammonia.
A merged mechanism in which the bridging water attacks the substrate, but with
His 320 acting as a general acid.
 Elimination mechanism to form a cyanic acid (O=C=NH) intermediate that
subsequently becomes hydrated (not depicted) to form carbamate.
 In all mechanisms, the carbamate spontaneously decomposes
Role of nickel in Urease

10. Rapid Urease Test (RUT)

11. The broth contains two pH buffers, urea, a very small amount of nutrients for the bacteria, and
the pH indicator phenol red. Phenol red turns yellow in an acidic environment and fuchsia in
an alkaline environment. If the urea in the broth is degraded and ammonia is produced, an
alkaline environment is created, and the media turns pink.
Urease broth Test

12. Fluorometric enzymatic assay of L-arginine
Molecular and Biomolecular Spectroscopy 2017
 Enzymatic method for Arg assay based on fluorometric monitoring of ammonia
 The selective analysis of ammonia (at 415 nm under excitation at 360 nm) is based on
reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium
 these conditions permit to avoid the reaction of OPA with any amino acid.
 A linearity range of the fluorometric arginase-urease-OPA method is from100 nM to 6
μМ with a limit of detection of 34nM Arg
 The method was used for the quantitative determination of Arg in the blood serum
 The proposed arginase- urease-OPA method being sensitive, economical, selective and
suitable for both routine and micro-volume formats
 It can be used in clinical diagnostics for the simultaneous determination of Arg as well
as urea and ammonia in serum samples.
Abstract

13. The reactions for Arg detection using arginase-urease-OPA-based method.
isoindole-1-thiol
o- Phthalaldehyde (OPA)

14. Preparation of OPA reagent
 0.2 g OPA + 5 mL 95% ethanol solution + 100 mL 0.1 M borate buffer, pH 10.
 The final reagent (14.2 mM OPA) was supplemented by sodium sulfite and stored in darkness
at room temperature until usage.
 The resulted OPA-ammonium product may be detected by spectrophotometry and fluorometry.
Ammonium ions calibration
 0.15 mL ammonium chloride solution with concentration from 0.005 mM to 1.25 mM in 50 mM
phosphate buffer, pH 7.0 (PB) was mixed in plastic tube with 3 mL OPA reagent, closed carefully
and heated at 60 °C for 15 min.
 As control, OPA reagent without ammonia (with PB) was used. Fluorescence emission value of
the tested sample was registered at 415 nm (under excitation at 360 nm).
The development of arginase-urease/OPAAssay
Urea calibration
 0.125 mL urea solution in PB with concentration from 0.005mM to 0.625mM was mixed in plastic
tubes with 0.010 mL urease solution and incubated during 20 min at 37 °C for complete conversion of
urea to ammonium ions.
The resulted ammonium ions, formed from urea, were detected after heating with 4 mL OPA
reagent.

15. Arg calibration
 0.1 mL Arg solution with concentration from 0.0025 mM to 0.625 mM in 30 mM Tris-HCl buffer,
pH 8.8 (TB) was mixed in plastic tube with 0.03 mL arginase solution in TB and was incubated
at 37 °C during 20 min for complete hydrolyses of Arg.
 Resulted solution was supplemented with an aliquot of 200 mM HCl (to achieve the pH 7.0,
optimal for urease type IX from Jack Beans) followed by addition of 0.020 mL urease solution.
 The reaction mixture was incubated during 20 min at 37 °C and the resulted ammonia was
detected fluorometrically after heating with 2.5 mL OPA , reaction mixture without Arg (but with
PB) was used.
Serum preparation and its pre-treatment
 The serum, sample of venous blood was incubated during 20 min at 20 °C and clotted fibrin was
removed by centrifugation (5 min, 4000 rpm).
 After 30 min incubation at 0 °C, the pellet of proteins was removed by centrifugation and
resulting supernatant was neutralized to pH 7.0 by adding 2 M Tris base.
 The neutralized protein-free solution (Sample 1), being ready for assay of Arg, urea and
ammonium, was kept at −20 °C till the usage.

16. where Ist and Ia – values of fluorescence emission of the standard
probe and tested Sample, respectively
Ca – concentration of endogenous ammonia in the Sample 1
Cst – concentration of standard ammonia solution
N – Dilution Factor of the tested Sample 1
where CArg,Ct
a andCe
a−concentrations of endogenous Arg, average contents of total
and endogenous ammonia in the tested sample, respectively

17. Arginine analysis in serum

18. 1 mM ammonia, interacted with OPA at 415 nm The concentrations of ammonia in final mixture (a), urea (b) and Arg (c)
Selectivity of the arginase/urease-OPA-based approach with various 0.5mM amino acids
A B
C

19. Real sample analysis of Arg level with different methods

20. Conclusion
 Fluorometric enzymatic is the selective, sensitive and low-cost methods for quantitative Arg
analysis
 A linearity range of the fluorometric arginase-urease-OPA method is from 100 nM to 6 μМ with a
limit of detection of 34 nM Arg
 Quantitative determination of Arg in the pooled sample of blood serum
 The reaction between ammonia and OPA (o-phthalaldehyde) can also proceed at room temp
 It would be useful for the simultaneous determination of Arg, urea and ammonia in blood
 predict a potential risk of metabolic disorders, hyperargininemia, autoimmune and other diseases