Creatinase

 

Creatinase or creatine amidinohydrolase (EC:3.5.3.3) catalyses the conversion of creatine and water to sarcosine and urea. This is a key step in the metabolic breakdown of creatinine by micro-organisms. Creatinase is also found in higher animals but its metabolic role is as yet not known.

The enzyme works as a homodimer, and is induced by choline chloride. Each monomer of creatinase has two clearly defined domains, a small N-terminal domain, and a large C-terminal domain. The C-terminal domain is a member of the MEROPS peptidase family M24 (clan MG), which share a common structural-fold, the "pita-bread" fold. The active site is located between the N- and C-terminal domains.

 

Reference Protein and Structure

Sequence
P38488 UniProt (3.5.3.3) IPR000994 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas putida (Bacteria) Uniprot
PDB
1chm - ENZYMATIC MECHANISM OF CREATINE AMIDINOHYDROLASE AS DEDUCED FROM CRYSTAL STRUCTURES (1.9 Å) PDBe PDBsum 1chm
Catalytic CATH Domains
3.90.230.10 CATHdb (see all for 1chm)
Cofactors
Water (1)
Click To Show Structure

Enzyme Reaction (EC:3.5.3.3)

creatine zwitterion
CHEBI:57947ChEBI
+
water
CHEBI:15377ChEBI
→
urea
CHEBI:16199ChEBI
+
sarcosine zwitterion
CHEBI:57433ChEBI

Enzyme Mechanism

Introduction

Histidine 232 abstracts a proton from an active site water molecule to create a hydroxide nucleophile. Glutamate 262 and 358 are negatively charged and break the resonance of the guanidinium group, this allows the hydroxide to attack at C1. Histidine 232 now donates its proton to N3 which leads to the C1-N3 bond breaking. Histidine 232 again abstracts a proton, this time from the hydroxide attached to C1, leaving the reaction products urea and sarcosine.

Catalytic Residues Roles

UniProt PDB* (1chm)
His232 His232(231)A The residue acts as a general base to the hydrolytic water molecule, a general acid to the intermediate, and then as a base once more in a heterolytic bimolecular elimination reaction, leading to the products. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Glu358, Glu262 Glu358(357)A, Glu262(261)A The residue's charged side chain breaks the resonance of the creatine gaunidinium group, allowing the hydroxyl to act as a nucleophile at the partial carbocation. hydrogen bond acceptor, electrostatic stabiliser
*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, intermediate formation, bimolecular elimination, intermediate collapse, intermediate terminated, native state of enzyme regenerated, inferred reaction step

References

  1. Coll M et al. (1990), J Mol Biol, 214, 597-610. Enzymatic mechanism of creatine amidinohydrolase as deduced from crystal structures. DOI:10.1016/0022-2836(90)90201-v. PMID:1696320.

Step 1. His232 deprotonates water, which initiates a nucleophilic attack on the guanidinium carbon of the substrate in an addition reaction.

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Catalytic Residues Roles

Residue Roles
Glu358(357)A hydrogen bond acceptor, electrostatic stabiliser
His232(231)A hydrogen bond acceptor
Glu262(261)A hydrogen bond acceptor, electrostatic stabiliser
His232(231)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation

Step 2. The methylated nitrogen of the guanidinium group deprotonates His232.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu358(357)A hydrogen bond acceptor
His232(231)A hydrogen bond donor
Glu262(261)A hydrogen bond acceptor
His232(231)A proton donor

Chemical Components

proton transfer, intermediate formation

Step 3. His232 deprotonates the hydroxyl group added, eliminating sarcosine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu358(357)A hydrogen bond acceptor
His232(231)A hydrogen bond acceptor
Glu262(261)A hydrogen bond acceptor
His232(231)A proton acceptor

Chemical Components

ingold: bimolecular elimination, intermediate collapse, intermediate terminated

Step 4. water deprotonates His232 in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His232(231)A hydrogen bond donor, proton donor

Chemical Components

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

Step 1. His232 deprotonates water, which initiates a nucleophilic attack on the guanidinium carbon of the substrate in an addition reaction.

Step 2. The methylated nitrogen of the guanidinium group deprotonates His232.

Step 3. His232 deprotonates the hydroxyl group added, eliminating sarcosine.

Step 4. water deprotonates His232 in an inferred return step.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Alex Gutteridge, Craig Porter