| Creatinase | |||||||||
|---|---|---|---|---|---|---|---|---|---|
 PDB image of creatinase enzyme structure as described by Hoeffken et al. 1993 | |||||||||
| Identifiers | |||||||||
| EC no. | 3.5.3.3 | ||||||||
| CAS no. | 37340-58-2 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDBPDBePDBsum | ||||||||
| Gene Ontology | AmiGO /QuickGO | ||||||||
| |||||||||
Inenzymology,creatinase (EC3.5.3.3), also known as creatine amidinohydrolase, is classified as ahydrolaseenzyme, acting on carbon-nitrogen bonds in linear amidines.[1] Specifically, this enzyme breaks the amidino C-N bond increatine, producingsarcosine andurea.[2] Creatinase activity has been described in several bacteria species, most notablyPseudomonas putida, where the enzyme plays a key role in the metabolism of creatine as anitrogen andcarbon source.[1]
Creatinase was first identified by Roche, Lacombe, & Girard in 1950[3] inPseudomonas eisenbergii andP. ovalis. It is produced by other bacterial genera includingBacillus,Flavobacterium,Micrococcus,Alcaligenes,Clostridium,Arthrobacter, andParacoccus, and is produced by other species ofPseudomonas as well.[1][4]
InP. putida, creatinase is coded for by thecreA gene and enables growth on creatine as the sole nitrogen source. Expression ofcreA is regulated by CahR, an AraC/GAT-Rregulator that activates gene expression in the presence of creatine.[5] This gene has also been cloned intoEscherichia coli.[2]
Creatinase is ahomodimeric enzyme with a calculatedmolecular mass of approximately 94,000 ± 2,000 Da.[6][2] Eachmonomer subunit contains 403 amino-acid residues split between two distinct structural domains.[2] The enzyme was purified and crystallized in 1976[6] after being extracted fromP. putida.
Creatinase has two domains:
Each noncovalently-associated monomer subunit consists of both domains. The subunits interact with each other through about 20hydrogen bonds and fourion pairs, providing stability to the dimer. The two domains in each monomer subunit are more loosely connected, with only six hydrogen bonds and one ion pair between them, and no intra- or intermoleculardisulfide bonds.[2]
Looking more closely at the two domains, the following structures were described in 1988:[7]
It has been suggested, following inhibition experiments, that a sulfhydryl group is located on or near the active site of the enzyme.[8]
Creatinasecatalyzes thechemical reaction
Creatine binds inside the β-half-barrel trough of the large C-terminal domain, forming hydrogen bonds between its amidino and carboxyl groups and the enzyme's amino-acid residues. A metal ion, eitherZn2+ orMn2+, is used to stabilize the substrate and polarize the amidino group.
The bound metal ion, plus a glutamate-histidine pair, activates and splits a water molecule from the surrounding solution. This generates ahydroxide ion within the active site.
The hydroxide ion joins with the carbon atom of the creatine's amidino group, creating a tetrahedral intermediate product. This intermediate product collapses and breaks the C-N bond, releasing urea. The remainder of the molecule rearranges to form sarcosine, and both products diffuse from the active site pocket.
For soil bacteria, creatinase allows for organisms to process the carbon and nitrogen that come from animal wastes, degrading creatine that is made in the kidney, liver, and pancreas and excreted through urine.[1] Animal tissues use creatine to buffer the charging of high-energy carriers during rapid ADP to ATP conversion, which creates1-methylhydantoin.[5] To avoid build up, animals excrete creatine andcreatinine in urine.
In humans, creatinase is used in enzymatic measurements of creatinine concentration for the diagnosis of renal and muscle diseases.[1] The enzyme catalyzes the second step of a coupled creatine assay, which is used to monitor the filtration rate of the glomeruli of the kidneys.[2]
Activity parameters pertaining to energetics and kinetics of the enzyme are as described below. The parameters were described through classic steady-state enzyme assays.[2][6]
| Parameter | Value | Notes |
|---|---|---|
| Km (creatine) | 1.33 mM | Moderate affinity |
| Kcat (creatine) | 0.246 s-1 | Slow turnover rate |
| Activation energy | 43 kJ/mol | Low-moderate |
| Specific activity | 15 U/mg | Low |
| Optimum pH | 8 | Stable between 6-8 |
| Optimum temperature | 30°C | 30 min half-life at 45°C |
