Creatinine Amidohydrolase (Creatininase) from Pseudomonas putida: ePurification and Some Properties1

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Abstract
Creatinine amidohydrolase [EC 3.5.2.-; creatininase] was purified in an overall yield of 11% from cell-free extract of Pseudomonas putida var. naraensis, strain C-83, by column chro-matographies on sarcosine-HM-Sepharose and DEAE-cellulose, and gel filtration on Sephadex G-200. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme catalyzed the reversible conversion of creatinine to creatine with an optimal pH of 7–9. Km values for creatinine and creatine were 26 mM and 0.13 m, respectively. The enzyme was also active toward glycocyamidine, though the reaction rate was quite low, but it was completely inert toward hydantoin and its derivatives. The molecular weight of the enzyme was estimated to be 175,000 by ultracentrifugal analysis and the subunit molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 23,000, suggesting that the enzyme is composed of eight subunit monomers. The isoelectric point was 4.7 as judged by iso-electric focusing experiments. The enzyme was markedly inactivated by heavy metal ions, iV-bromosuccinimide, ethoxyformic anhydride, and dye-sensitized photooxidation, and also partially by metal chelators. The enzyme was found to contain about one gram atom of zinc per subunit monomer. The metal-free, inactive enzyme was prepared and could be reactivated by the addition of Mn2+, Co2+, Mg2+, Fe2+, Ni2+, and Zn2+ in that order of decreasing effectiveness. These results indicate that metal is intimately involved in the creatininase activity of P. putida.