title: Localization and functional analysis of the substrate specificity/catalytic domains of human M-form and P-form phenol sulfotransferases.
abstract: Human monoamine (M)-form and simple phenol (P)-form phenol sulfotransferases (PSTs), which are greater than 93% identical in their primary sequences, were used as models for investigating the structural determinants responsible for their distinct substrate specificity and other enzymatic properties. A series of chimeric PSTs were constructed by reciprocal exchanges of DNA segments between cDNAs encoding M-form and P-form PSTs. Functional characterization of the recombinant wild-type M-form, P-form, and chimeric PSTs expressed in Escherichia coli and purified to homogeneity revealed that internal domain-spanning amino acid residues 84-148 contain the structural determinants for the substrate specificity of either M-form or P-form PST. Data on the kinetic constants (Km, Vmax, and Vmax/Km) further showed the differential roles of the two highly variable regions (Region I spanning amino acid residues 84-89 and Region II spanning amino acid residues 143-148) in substrate binding, catalysis, and sensitivity to the inhibition by 2,6-dichloro-4-nitrophenol. In contrast to the differential sulfotransferase activities of M-form and P-form PSTs toward dopamine and p-nitrophenol, the Dopa/tyrosine sulfotransferase activities were found to be restricted to M-form, but not P-form, PST. Furthermore, the variable Region II of M-form PST appeared to play a predominant role in determining the Dopa/tyrosine sulfotransferase activities of chimeric PSTs. Kinetic studies indicated the role of manganese ions in dramatically enhancing the binding of D-p-tyrosine to wild-type M-form PST. Taken together, these results pinpoint unequivocally the sequence encompassing amino acid residues 84-148 to be the substrate specificity/catalytic domain of both M-form and P-form PSTs and indicate the importance of the variable Regions I and II in determining their distinct enzymatic properties.
authors.affiliation: Department of Biochemistry, University of Texas Health Center, Tyler, Texas 75710, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Sakakibara
authors.forename: Y
authors.initials: Y
authors.lastname: Takami
authors.forename: T
authors.initials: T
authors.lastname: Nakayama
authors.forename: M
authors.initials: M
authors.lastname: Suiko
authors.forename: M C
authors.initials: MC
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D015239:Arylsulfotransferase / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D004298:Dopamine / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D009596:Nitrophenols; D011993:Recombinant Fusion Proteins; D008345:Manganese; C015802:2,6-dichloro-4-nitrophenol; D015239:Arylsulfotransferase; D004298:Dopamine; C024836:4-nitrophenol