Urease

 

Ureases hydrolyse urea into ammonia and carbamate. Ureases have been isolated from a wide range of bacteria, fungi and higher plants where it allows the organism to use urea as a nitrogen source. Ureases uses an almost unique bi-nickel catalytic centre which is liganded by a carbamylated lysine.

The mechanism of this enzyme has been subject to debate since the early 1920s and the precise steps in catalysis remain unclear [PMID:20471401].

 

Reference Protein and Structure

Sequences
P18316 UniProt (3.5.1.5)
P18315 UniProt (3.5.1.5)
P18314 UniProt (3.5.1.5) IPR005848 (Sequence Homologues) (PDB Homologues)
Biological species
[Enterobacter] aerogenes (Bacteria) Uniprot
PDB
1fwj - KLEBSIELLA AEROGENES UREASE, NATIVE (2.2 Å) PDBe PDBsum 1fwj
Catalytic CATH Domains
3.20.20.140 CATHdb (see all for 1fwj)
Cofactors
Nickel(2+) (2), Water (3) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.5.1.5)

urea
CHEBI:16199ChEBI
+
water
CHEBI:15377ChEBI
→
carbon dioxide
CHEBI:16526ChEBI
+
ammonia
CHEBI:16134ChEBI

Enzyme Mechanism

Introduction

The active site contains two nickels. Carbamylated lysine 217 provides an oxygen ligand to each nickel. One nickel ion is coordinated by three ligands: two histidines (246 and 272) and the carbamylated lysine 217 (with low occupancy of a fourth ligand) and the second is coordinated by five ligands: two histidines (136 and 134), aspartate 360, a water and the carbamylated lysine 217. Urea enters the active site and ligates to Ni-1 to complete its tetrahedral coordination. It also forms a hydrogen bond to histidine 219 which polarises the urea carbonyl. Histidine 320 acts as a general base and abstracts a proton from the Ni-2 water ligand, the resulting hydroxide ligand of Ni-2 then attacks the urea carbonyl carbon. The resulting tetrahedral intermediate decomposes, with the assistance of an unidentified general acid, to ammonia and Ni bound carbamylate which then dissociates.

Catalytic Residues Roles

UniProt PDB* (1fwj)
Lys217 (ptm) Kcx217C (ptm) Post-translationally modified lysine residue. Acts as a bridging ligand between the two Ni(II) ions. activator, metal ligand
Asp360 Asp360C Forms part of the nickel 1 binding site, also helps activate His320. metal ligand
His134, His136 His134C, His136C Forms part of the nickel 1 binding site. metal ligand
His246, His272 His246C, His272C Forms part of the nickel 2 binding site. metal ligand
His320, His219 His320C, His219C Acts as a general acid/base. proton acceptor, proton donor
Asp221 Asp221C Acts as a general acid/base, activating His219 . activator, proton acceptor, proton donor
Arg336 Arg336C Helps stabilise the charge in the active site, activating both His320 and Asp221. activator
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic addition, deamination, unimolecular elimination by the conjugate base, reaction occurs outside the enzyme, intramolecular elimination

References

  1. Balasubramanian A et al. (2010), J Mol Biol, 400, 274-283. Crystal Structure of the First Plant Urease from Jack Bean: 83 Years of Journey from Its First Crystal to Molecular Structure. DOI:10.1016/j.jmb.2010.05.009. PMID:20471401.
  2. Roberts BP et al. (2012), J Am Chem Soc, 134, 9934-9937. Wide-Open Flaps Are Key to Urease Activity. DOI:10.1021/ja3043239. PMID:22670767.
  3. Carlsson H et al. (2010), Bioinorg Chem Appl, 2010, 1-8. Computational Modeling of the Mechanism of Urease. DOI:10.1155/2010/364891. PMID:20886006.
  4. Krajewska B (2009), J Mol Catal B Enzym, 59, 9-21. Ureases I. Functional, catalytic and kinetic properties: A review. DOI:10.1016/j.molcatb.2009.01.003.
  5. Barrios AM et al. (2000), J Am Chem Soc, 122, 9172-9177. Interaction of Urea with a Hydroxide-Bridged Dinuclear Nickel Center:  An Alternative Model for the Mechanism of Urease. DOI:10.1021/ja000202v.
  6. Pearson MA et al. (2000), Biochemistry, 39, 8575-8584. Kinetic and Structural Characterization of Urease Active Site Variants†,‡. DOI:10.1021/bi000613o. PMID:10913264.
  7. Pearson MA et al. (1998), Biochemistry, 37, 6214-6220. Chemical Rescue ofKlebsiella aerogenesUrease Variants Lacking the Carbamylated-Lysine Nickel Ligand†,‡. DOI:10.1021/bi980021u. PMID:9558361.
  8. Park IS et al. (1996), J Biol Chem, 271, 18632-18637. Characterization of the Mononickel Metallocenter in H134A Mutant Urease. DOI:10.1074/jbc.271.31.18632. PMID:8702515.
  9. Jabri E et al. (1996), Biochemistry, 35, 10616-10626. Structures of theKlebsiella aerogenesUrease Apoenzyme and Two Active-Site Mutants†,‡. DOI:10.1021/bi960424z. PMID:8718850.
  10. Jabri E et al. (1995), Science, 268, 998-1004. The crystal structure of urease from Klebsiella aerogenes. DOI:10.1126/science.7754395. PMID:7754395.
  11. Park IS et al. (1993), Protein Sci, 2, 1034-1041. Site-directed mutagenesis ofKlebsiella aerogenesurease: Identification of histidine residues that appear to function in nickel ligation, substrate binding, and catalysis. DOI:10.1002/pro.5560020616. PMID:8318888.

Step 1. Asp221 deprotonates His219, which in turn deprotonates the nickel bound urea, forming the active conformer for this mechanism.

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Catalytic Residues Roles

Residue Roles
His134C metal ligand
His136C metal ligand
Asp360C metal ligand
His246C metal ligand
His272C metal ligand
Kcx217C (ptm) metal ligand
Kcx217C (ptm) activator
Arg336C activator
His219C proton acceptor, proton donor
Asp221C proton acceptor
His219C proton relay

Chemical Components

proton transfer

Step 2. His320 deprotonates nickel bound water, which initiates a nucleophilic attack on the nickel bound urea in an addition reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Kcx217C (ptm) activator, metal ligand
His134C metal ligand
His136C metal ligand
Asp360C metal ligand
His246C metal ligand
His272C metal ligand
His219C proton acceptor, proton donor
His320C proton acceptor
Asp221C proton donor
His219C proton relay

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 3. The tetrahedral intermediate collapses, eliminating ammonia with concomitant deprotonation of His320.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His134C metal ligand
His136C metal ligand
Asp360C metal ligand
His246C metal ligand
His272C metal ligand
Kcx217C (ptm) metal ligand
Asp221C activator
Arg336C activator
Kcx217C (ptm) activator
His320C proton donor

Chemical Components

deamination, ingold: unimolecular elimination by the conjugate base

Step 4. Carbamate dissociates from the active site and is spontaneously converted to carbon dioxide and a second molecule of ammonia

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, ingold: intramolecular elimination
Download: Image, Marvin File

Step 1. Asp221 deprotonates His219, which in turn deprotonates the nickel bound urea, forming the active conformer for this mechanism.

Step 2. His320 deprotonates nickel bound water, which initiates a nucleophilic attack on the nickel bound urea in an addition reaction.

Step 3. The tetrahedral intermediate collapses, eliminating ammonia with concomitant deprotonation of His320.

Step 4. Carbamate dissociates from the active site and is spontaneously converted to carbon dioxide and a second molecule of ammonia

Products.

Introduction

A third proposal is the elimination reaction from Barios and Lippard [PMID:11300826] has also been proposed on the observation of cyanic intermediates. Theoretical work by Estiu and Merz [PMID:16584179, PMID:17676790] suggests that the elimination pathway may occur in competition with the more traditionally proposed mechanisms.

Catalytic Residues Roles

UniProt PDB* (1fwj)
Lys217 (ptm) Kcx217C (ptm) Post-translationally modified lysine residue. Acts as a bridging ligand between the two Ni(II) ions. metal ligand
Asp360 Asp360C Forms part of the nickel 1 binding site, also helps activate His320. activator, metal ligand
His134, His136 His134C, His136C Forms part of the nickel 1 binding site. metal ligand
His246, His272 His246C, His272C Forms part of the nickel 2 binding site. metal ligand
His320, His219 His320C, His219C Acts as a general acid/base. proton acceptor, proton donor
Asp221 Asp221C Acts as a general acid/base, deprotonating His219 to activate it. It later donates this proton to His320. proton acceptor, proton donor
Arg336 Arg336C Helps stabilise the charge in the active site, activating both His320 and Asp221. activator
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

reaction occurs outside the enzyme, bimolecular nucleophilic addition, inferred reaction step, intramolecular elimination, native state of enzyme regenerated

References

  1. Barrios AM et al. (2001), Inorg Chem, 40, 1250-1255. Decomposition of Alkyl-Substituted Urea Molecules at a Hydroxide-Bridged Dinickel Center. DOI:10.1021/ic000933w. PMID:11300826.
  2. Roberts BP et al. (2012), J Am Chem Soc, 134, 9934-9937. Wide-Open Flaps Are Key to Urease Activity. DOI:10.1021/ja3043239. PMID:22670767.
  3. Krajewska B (2009), J Mol Catal B Enzym, 59, 9-21. Ureases I. Functional, catalytic and kinetic properties: A review. DOI:10.1016/j.molcatb.2009.01.003.
  4. Estiu G et al. (2007), J Phys Chem B, 111, 10263-10274. Competitive Hydrolytic and Elimination Mechanisms in the Urease Catalyzed Decomposition of Urea. DOI:10.1021/jp072323o. PMID:17676790.
  5. Estiu G et al. (2006), Biochemistry, 45, 4429-4443. Catalyzed Decomposition of Urea. Molecular Dynamics Simulations of the Binding of Urea to Urease†. DOI:10.1021/bi052020p. PMID:16584179.
  6. Park IS et al. (1996), J Biol Chem, 271, 18632-18637. Characterization of the Mononickel Metallocenter in H134A Mutant Urease. DOI:10.1074/jbc.271.31.18632. PMID:8702515.

Step 1. Asp221 deprotonates His219, which in turn abstracts a proton from the Ni(II) bound urea, forming a negatively charged intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His134C metal ligand
His136C metal ligand
Asp360C metal ligand
His246C metal ligand
His272C metal ligand
Arg336C activator
Kcx217C (ptm) metal ligand
Asp221C proton acceptor
His219C proton acceptor, proton donor, proton relay

Chemical Components

Step 2. His320 deprotonats Asp221.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His134C metal ligand
His136C metal ligand
Asp360C metal ligand
His246C metal ligand
His272C metal ligand
Arg336C activator
Asp360C activator
Kcx217C (ptm) metal ligand
Asp221C proton donor
His320C proton acceptor

Chemical Components

Step 3. The negatively charged intermediate collapses, eliminating ammoinia, which abstracts the proton from His320.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His134C metal ligand
His136C metal ligand
Asp360C metal ligand
His246C metal ligand
His272C metal ligand
Arg336C activator
Asp360C activator
Kcx217C (ptm) metal ligand
His320C proton donor

Chemical Components

Step 4. Cyanic acid is the product of this enzyme mechanism, thus it is likely that the final hydrolysis occurs outside of the active site.

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Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, ingold: bimolecular nucleophilic addition, inferred reaction step

Step 5. Ammonia is eliminated in the final non-enzyme catalysed step of the reaction.

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Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, ingold: intramolecular elimination, inferred reaction step

Step 6. Inferred step to regenerate the enzyme's active site. Two water molecules displace the product to form the ground state. It is unclear how the His219 returns to it's native state, we have represented a water facilitated tautomerisation reaction here.

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Catalytic Residues Roles

Residue Roles
His134C metal ligand
His136C metal ligand
Asp360C metal ligand
His246C metal ligand
His272C metal ligand
Kcx217C (ptm) metal ligand
His219C proton relay, proton acceptor, proton donor

Chemical Components

inferred reaction step, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Asp221 deprotonates His219, which in turn abstracts a proton from the Ni(II) bound urea, forming a negatively charged intermediate.

Step 2. His320 deprotonats Asp221.

Step 3. The negatively charged intermediate collapses, eliminating ammoinia, which abstracts the proton from His320.

Step 4. Cyanic acid is the product of this enzyme mechanism, thus it is likely that the final hydrolysis occurs outside of the active site.

Step 5. Ammonia is eliminated in the final non-enzyme catalysed step of the reaction.

Step 6. Inferred step to regenerate the enzyme's active site. Two water molecules displace the product to form the ground state. It is unclear how the His219 returns to it's native state, we have represented a water facilitated tautomerisation reaction here.

Products.

Introduction

Proposed by Benini et al. for Bacillus pasteurii enzyme, urea binds in a bidentate manner with its carbonyl oxygen bound to Ni1 and one of the amino group bound to Ni2, thus replacing three water moieties, leaving only the bridging hydroxide. This hydroxide attacks urea to give the tetrahedral transition state leading to formation of ammonia and carbamate.

Catalytic Residues Roles

UniProt PDB* (1fwj)
Lys217 (ptm) Kcx217C (ptm) Post-translationally modified lysine residue. Acts as a bridging ligand between the two Ni(II) ions. activator, metal ligand
Asp360 Asp360C Forms part of the nickel 1 binding site, also helps activate His320. metal ligand
His320 His320C Acts as a general acid/base. proton acceptor, proton donor
His134, His136 His134C, His136C Forms part of the nickel 1 binding site. metal ligand
His246, His272 His246C, His272C Forms part of the nickel 2 binding site. metal ligand
His219 His219C Has no assigned role in this mechanism.
Asp221, Arg336 Asp221C, Arg336C Activates His320. activator
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, unimolecular elimination by the conjugate base, inferred reaction step, reaction occurs outside the enzyme, intramolecular elimination

References

  1. Benini S et al. (2001), J Biol Inorg Chem, 6, 778-790. Structure-based rationalization of urease inhibition by phosphate: novel insights into the enzyme mechanism. DOI:10.1007/s007750100254. PMID:11713685.
  2. Roberts BP et al. (2012), J Am Chem Soc, 134, 9934-9937. Wide-Open Flaps Are Key to Urease Activity. DOI:10.1021/ja3043239. PMID:22670767.
  3. Carlsson H et al. (2010), Bioinorg Chem Appl, 2010, 1-8. Computational Modeling of the Mechanism of Urease. DOI:10.1155/2010/364891. PMID:20886006.
  4. Krajewska B (2009), J Mol Catal B Enzym, 59, 9-21. Ureases I. Functional, catalytic and kinetic properties: A review. DOI:10.1016/j.molcatb.2009.01.003.
  5. Benini S et al. (2004), J Am Chem Soc, 126, 3714-3715. Molecular Details of Urease Inhibition by Boric Acid:  Insights into the Catalytic Mechanism. DOI:10.1021/ja049618p. PMID:15038715.
  6. Benini S et al. (1999), Structure, 7, 205-216. A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels. DOI:10.1016/s0969-2126(99)80026-4. PMID:10368287.
  7. Pearson MA et al. (1997), Biochemistry, 36, 8164-8172. Structures of Cys319 Variants and Acetohydroxamate-InhibitedKlebsiella aerogenesUrease†,‡. DOI:10.1021/bi970514j. PMID:9201965.
  8. Park IS et al. (1996), J Biol Chem, 271, 18632-18637. Characterization of the Mononickel Metallocenter in H134A Mutant Urease. DOI:10.1074/jbc.271.31.18632. PMID:8702515.

Step 1. Urea binds in a bindentate manner, displacing the active site water molecules. The bridging hydroxide group initiates a nucleophillic attack on the carbamyl carbon, forming a tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg336C activator
Kcx217C (ptm) activator, metal ligand
His134C metal ligand
His136C metal ligand
Asp360C metal ligand
His246C metal ligand
His272C metal ligand

Chemical Components

proton transfer

Step 2. The unbound NH2 group of the tetrahedral intermediate deprotonates His320.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His134C metal ligand
His136C metal ligand
Asp360C metal ligand
His246C metal ligand
His272C metal ligand
Kcx217C (ptm) activator, metal ligand
Asp221C activator
Arg336C activator
His320C proton donor

Chemical Components

proton transfer

Step 3. The tetrahedral intermediate collapses, eliminating ammonia. The products then dissociate from the active site, replaced by water molecules.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His134C metal ligand
His136C metal ligand
Asp360C metal ligand
His246C metal ligand
His272C metal ligand
Kcx217C (ptm) activator, metal ligand

Chemical Components

ingold: unimolecular elimination by the conjugate base

Step 4. In an inferred step, His320 abstracts a proton from one of the incoming water molecules to regenerate the enzyme's active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His134C metal ligand
His136C metal ligand
Asp360C metal ligand
His246C metal ligand
His272C metal ligand
Kcx217C (ptm) metal ligand
Asp221C activator
His320C proton acceptor

Chemical Components

inferred reaction step

Step 5. The carbamate product rearranges outside of the active site to produce carbon dioxide and ammonia.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, ingold: intramolecular elimination
Download: Image, Marvin File

Step 1. Urea binds in a bindentate manner, displacing the active site water molecules. The bridging hydroxide group initiates a nucleophillic attack on the carbamyl carbon, forming a tetrahedral intermediate.

Step 2. The unbound NH2 group of the tetrahedral intermediate deprotonates His320.

Step 3. The tetrahedral intermediate collapses, eliminating ammonia. The products then dissociate from the active site, replaced by water molecules.

Step 4. In an inferred step, His320 abstracts a proton from one of the incoming water molecules to regenerate the enzyme's active site.

Step 5. The carbamate product rearranges outside of the active site to produce carbon dioxide and ammonia.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Anna Waters, Craig Porter