A Bacterial Halidohydrolase. Its Purification, Some Properties and its Modification by Specific Amino Acid Reagents

1 The purification of a halidohydrolase from cell-free extracts of Pseudomonas dehalogenans NCIB 9061 grown on glucose and monochloroacetate as carbon source, is described. 2 This enzyme catalyses the hydrolysis of some 2-halogen substituents on aliphatic carboxylic acids, releasing a halide ion and a hydrogen ion and causing inversion of the configuration. 3 The molecular weight as determined by chromatography on Sephadex G-100 was about 15000. 4 The optimum activity was at pH 9.4. Activity was rapidly and irreversibly lost below pH 6.0. 5 The amino acid composition of the purified enzyme in its native state and after photooxidation in the presence of methylene blue is presented. 6 The variation of the kinetic parameters, V and Km, between pH 7.0 and pH 10.0 suggest that a group with a pKa of about 7.8 is involved in its base form in the catalytic reaction. 7 The enzyme is rapidly inactivated by reaction with iodine and upon photooxidation in the presence of methylene blue, but the effect of thiol reagents suggests that a thiol group is not directly responsible for catalysis. 8 It is suggested that a histidine residue is implicated in the catalytic reaction and a mechanism is proposed which is consistent with the experimental results.