ISOLATION AND CHARACTERIZATION OF A CYANIDE DIHYDRATASE FROM BACILLUS-PUMILUS C1

A cyanide-degrading enzyme from Bacillus pumilus C1 has been purified and characterized. This enzyme consisted of three polypeptides of 45.6 , 44.6, and 41.2 kDa; the molecular mass by gel filtration was 417 kDa . Electron microscopy revealed a multimeric, rod-shaped protein approx imately 9 by 50 nm. Cyanide was rapidly degraded to formate and ammoni a. Enzyme activity was optimal at 37-degrees-C and pH 7.8 to 8.0. Acti vity was enhanced by Sc3+, Cr3+, Fe3+, and Tb3+; enhancement was indep endent of metal ion concentration at concentrations above 5 muM. Rever sible enhancement of enzymatic activity by azide was maximal at 4.5 mM azide and increased with time. No activity was recorded with the cyan ide substrate analogs CNO-, SCN-, CH3CN, and N3- and the possible degr adation intermediate HCONH2. Kinetic studies indicated a K(m) of 2.56 +/- 0.48 mM for cyanide and a V(max) of 88.03 +/- 4.67 mmol of cyanide per min/mg/liter. The K(m) increased approximately twofold in the pre sence of 10 muM Cr3+ to 5.28 +/- 0.38 mM for cyanide, and the V(max) i ncreased to 197.11 +/- 8.51 mmol of cyanide per min/mg/liter. We propo se naming this enzyme cyanide dihydratase.