title: Kinetic mechanism and identification of the active site tyrosine residue in Enterobacter amnigenus arylsulfate sulfotransferase.
abstract: Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of a sulfate group from a phenyl sulfate ester to a phenolic acceptor. The kinetic mechanism of Enterobacter amnigenus ASST was determined. Plots of 1/v versus 1/[substrate (A)] at different fixed substrate (B) concentrations gave a series of parallel lines. One of the reaction products, p-nitrophenol, inhibited the enzyme noncompetitively with respect to p-nitrophenyl sulfate, but competitively to alpha-naphthol. These results correspond to a ping pong bi bi mechanism. By site-directed mutagenesis, we substituted each conserved tyrosine residue with phenylalanine. Among the mutants, Y123F showed severely reduced catalytic activity. We conclude that Tyr 123 is an essential active site residue. A mechanistic hypothesis is presented to account for these observations.
authors.affiliation: College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742, Korea.
authors.forename: A R
authors.initials: AR
authors.lastname: Kwon
authors.forename: H J
authors.initials: HJ
authors.lastname: Yun
authors.forename: E C
authors.initials: EC
authors.lastname: Choi
mesh_terms: D019943:Amino Acid Substitution; D015239:Arylsulfotransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D004754:Enterobacter / Q000201:enzymology*; D004926:Escherichia coli; D005246:Feedback; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine
chemical_list: D009596:Nitrophenols; D011994:Recombinant Proteins; D014443:Tyrosine; D015239:Arylsulfotransferase; C090707:arylsulfate sulfotransferase; C024836:4-nitrophenol