Decomposition of Urea with Urease

kep Decomposition of Urea with Urease

Demonstration Experiment on Video

Decomposition of Urea with Urease

Objectives: Enzyme Catalysis, Substrate Specifity, Competitive Inhibition

Peter Keusch

German version

Chemicals:
urea (M = 60.06 g / mol)
thiourea (M = 76.13 g / mol)
urease
Phenolphthalein solution 1% in ethanol (Merck)

Glass wares:
3 conical measures, graduated, glass base, 350 mL
3 glass stirring rods
3 snap-cap vials 10 mL
3 snap-cap vials 20 mL

Hazards and safety precautions:

Thiourea is toxic. Known animal carcinogen and probable human carcinogen. May cause irreversible effects. May affect fertility. May be fatal if swallowed. May cause allergic skin reaction. May cause skin ulcers, liver damage.

Handle as a carcinogen. Gloves, safety glasses, good ventilation. Protect against spills and the spread of dust.

Preparation of the solutions

Urea solution: 2.4 g of urea in 200 mL of dist. H₂0
Urea/thiourea solution: 2.4 g of urea + 3 g of thiourea in 200 mL of dist. H₂0
Thiourea solution: 3 g of thiourea in 200 mL of dist. H₂0
Urease suspension: 60 mg of urease in 30 mL of dist. H₂0

Experimental procedure:

Three conical measures are set up as described below:
Conical measure A: 200 mL of urea solution
Conical measure B: 200 mL of urea / thiourea solution
Conical measure C: 200 mL of thiourea solution

2 mL of Phenolphthalein solution and 10 ml of aqueous urease suspension are added to the solutions in A, B amd C while stirring.

Results:

The urea solution in conical measure A turns red. The color becomes more intense within a few minutes. After a few minutes the urea / thiourea mixture in conical measure B starts to turn a light red.

The aqueous solution of thiourea in conical measure C remains colorless.

Video clip (Download RealPlayer .rm file)

Discussion:

Urease is a nickel metallo enzyme that catalyzes the degredation of urea to ammonia and carbamine acid. The latter compound decomposes to generate a second molecule of ammonia and carbon dioxide.

The crystal structure of the active centre of urease contains probably two simple coordinated water molecules and a bridging OH group. The substrate binding site of urease is perfect constructed. The specificity of the enzyme is closely related to the shape of its active centre.

Fig.1: Active centre of urease

The supposed mechanism (Fig. 2) is a cooperative interaction between the two Ni ions.

· One Ni ion (Ni1) acts as a Lewis acid. The metal ion polarizes the carbonyl group of urea and activates it toward nucleophilic attack. In the process two H₂O molecules are replaced by urea. The other Ni ion (Ni2) binds a hydroxide ion.
· The OH^- ion attacks the partially positive carbonyl carbon of the urea molecule. One of the two NH₂ groups of urea is protonated. Histidine is postulated to be the acid.

The reactions result in the formation of ammonia abd carbamine acid.

Fig.2: Mechanism of the decompostion of urea

Carbamine acid decomposes spontaneously to form ammonia and carbon dioxide (1)

The aqueous solution turns phenolphthalein red due to the presence of hydroxide ions.

Urease is absolutely specific for its substrate urea, and structural analogues (e.g. thiourea) are not degraded by the enzyme.

If urease is added to a mixture of urea and thiourea the activity of the enzyme is strongly diminished. Like urea thiourea is bound to the active centre of the enzyme. Thus the active site of the enzyme is temporarily blocked by the 'false substrate'. The inhibition is called competitive because urea and thiourea compete on equal terms for the binding site of urease. Characteristically increasing the concentration of substrate reduces the effect of the inhibitor. Inhibition can be overcome.

Fig.1 and Fig.2 taken from Bioanorganische Chemie I (Eisen, Molybdän und Nickel (Uni Hamburg)

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