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The extracellular cytosine deaminase (EC 3.5.4.1) from Chromobacterium violaceum YK 391 was purified 264.7-fold with an overall yield of 14.3%. The enzyme was for the first time homogeneous by the criteria of polyacrylamide gel electrophoresis performed in the absence and in the presence of sodium dodecyl sulfate. The molecular weight of the purified enzyme was estimated to be about 156 kDa. The enzyme consisted of two identical subunits of approximate molecular weight 78 kDa. The isoelectric point of the enzyme was pH 5.55. The enzyme had a pH optimum of 7.5 and a temperature optimum of around 40 to 45℃. Besides cytosine, the enzyme deaminated 5-fluorocytosine, cytidine, 5-methylcytosilie, and 6-azacytosine, but not 5-azacytosine. The extracellular cytosine deaminase is believed to he unique because it was active not only on cytosine but also on cytidine. The apparent K_m values for cytosine, 5-fluorocytosine, cytidine, and 5methylcytosine were determined to he 1.55 mM, 5.52 mM, 10.4 mM, and 67.2 mM, respectively. The enzyme activity was strongly inhibited by heavy metal ions such as Fe^2+. Pb^2+, Cd^2+, Zn^2+, Hg^2+, and Cu^2+ at 1 mM, and completely by α,α^1- dipyridyl, and ρ-chloromercuribenzoate at 1 mM, and weakly inhibited by I mM ο- phenanthroline. The enzyme activity was not affected by various nucleosides and nucleotides.
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