Creatinase

Different possibilities for conducting enzymatic assays on microchip platforms including pre-, on- or post-column reactions have been reviewed [156]. An enzymatic assay (employing creatininase, creatinase and sarcosine oxidase) has also been developed in a microchip for analysing renal marks such as creatinine and creatine as well as p-aminohippuric acid and uric acid [157]. 

Rowe DJ, Omar H, Barratt SL, Biggs P. An evaluation of blood creatinine measurement by creatinase on the NOVA M7 blood gas analyser. Clin Chim Acta 2001 307 23-5. 

Creatine and creatinine Creatininase, creatinase, and sarcosine oxidase 30 mM... 

Beuth, B., Niefind, K. and Schomburg, D. (2003). Crystal structure of creatinase from Pseudomonas putida a novel fold and a case of convergent evolution. Journal of Molecular Biology, 332, 287-301. 

Creatinase catalyzes the hydrolysis of creatine to N-methyl-glycine and urea by a mechanism in which water is activated for attack on substrate guanidinium carbon by the imidazole of His acting as general base [66]. 

Departure of urea leaving group during turnover of creatine by creatinase is facilitated by proton donation by the imidazolium group of His232, the same residue that extracted the proton from water to promote nucleophilic attack [66]. 

Nucleophilic Addition and Amide Bond Fission in Creatinase... 

The first example describes the attack of a nucleophile on a carbonyl-type carbon atom. This reaction is the first step in the creatinase catalyzed cleavage of the amide bond of creatine, which eventually leads to the products urea and sarcosine [79]. 

Natural substrate (guanidinium) and inhibitors (carbamoyl) differ by the presence or absence of a positive charge on this part of the molecule. Thus, addition of water (as OH ) to the carbamoyl moiety and hydrolysis of the inhibitor would result in the development of charge rather than in its dispersal (as for the natural substrate ) in the partially hydrophobic environment of the active site. This explains why carbamoyl sarcosine acts as inhibitor for creatinase. In succinamic acid, the central NCH3 is replaced by CH2 and the proton transfer reaction (step 3) cannot occur. 

The structural studies on creatinase illustrate a problem which, although not directly related to the discussion above, is worth discussing because its importance is... 

The author is grateful to Prof. Dr. R. Huber (Martinsried) for making structural data on the enzyme creatinase available. Special thanks go to Prof. Dr. G.A. Jeffrey (Pittsburgh), Dr. T. L. Poulos (Maryland) and Prof. Dr. A. Camerman (Seattle), who supplied results and material prior to publication. Sincere thanks go to my colleagues and friends in BASF and at various academic institutes for many stimulating and fruitful discussions, in particular to Dr. H. J. Bohm (BASF), who also helped with some of the computer simulations. 

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