title: Reaction mechanism of the Co2+-activated multifunctional bromoperoxidase-esterase from Pseudomonas putida IF-3.
abstract: The reaction mechanism of the Co2+-activated bromoperoxidase-esterase of Pseudomonas putida IF-3 was studied. Site-directed mutagenesis suggested that the serine residue of the catalytic triad conserved in serine hydrolases participates in the bromination and ester hydrolysis reactions. The enzyme released a trace amount of free peracetic acid depending on the concentration of H2O2, which had been considered the intermediate in the reaction of nonmetal haloperoxidases to oxidize halide ions to hypohalous acid. However, the formation of free peracetic acid could not explain the enzyme activation effect by Co2+ ions which completely depleted the free peracetic acid. In addition, the kcat value of the enzymatic bromination was 900-fold higher than the rate constant of free peracetic acid-mediated bromination. Those results strongly suggested that the peracetic acid-like intermediate formed at the catalytic site is the true intermediate and that the formation of free peracetic acid is only a minor reaction involving the enzyme. We propose the possible reaction mechanism of this multifunctional enzyme based on these findings.
authors.affiliation: Biotechnology Research Center, Toyama Prefectural University, Kurokawa 5180, Kosugi, Toyama, 939-0398, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Kawanami
authors.forename: Masao
authors.initials: M
authors.lastname: Miyakoshi
authors.forename: Tohru
authors.initials: T
authors.lastname: Dairi
authors.forename: Nobuya
authors.initials: N
authors.lastname: Itoh
mesh_terms: D003035:Cobalt / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D010463:Peracetic Acid / Q000378:metabolism; D010544:Peroxidases / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology*; D013440:Sulfides / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D013440:Sulfides; D003035:Cobalt; D010544:Peroxidases; C427221:cobalt(II)-activated bromoperoxidase-esterase; D010463:Peracetic Acid