title: Hydroxylase activity of Met471Cys tyramine beta-monooxygenase.
abstract: A series of mutations was targeted at the methionine residue, Met471, coordinating the Cu(M) site of tyramine beta-monooxygenase (TbetaM). The methionine ligand at Cu(M) is believed to be key to dioxygen activation and the hydroxylation chemistry of the copper monooxygenases. The reactivity and copper binding properties of three TbetaM mutants, Met471Asp, Met471Cys, and Met471His, were examined. All three mutants show similar metal binding affinities to wild type TbetaM in the oxidized enzyme forms. EPR spectroscopy suggests that the Cu(II) coordination geometry is identical to that of the WT enzyme. However, substrate hydroxylation was observed for the reaction of tyramine solely with Met471Cys TbetaM. Met471Cys TbetaM provides the first example of an active mutant directed at the Cu(M) site of this class of hydroxylases. The reactivity and altered kinetics of the Met471Cys mutant further highlight the central role of the methionine residue in the enzyme mechanism. The sole ability of the cysteine residue to support activity among the series of alternate amino acids investigated is relevant to theoretical and biomimetic investigations of dioxygen activation at mononuclear copper centers.
authors: Department of Chemistry, University of California, Berkeley, California 94720-3220, USA.
authors: Corinna R
authors: CR
authors: Hess
authors: Zinian
authors: Z
authors: Wu
authors: Adora
authors: A
authors: Ng
authors: Erin E
authors: EE
authors: Gray
authors: Michele A
authors: MA
authors: McGuirl
authors: Judith P
authors: JP
authors: Klinman
mesh_terms: D000818:Animals; D002384:Catalysis; D003300:Copper / Q000378:metabolism; D004330:Drosophila / Q000201:enzymology / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008715:Methionine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship
chemical_list: D003300:Copper; D008715:Methionine; D006899:Mixed Function Oxygenases; C023205:tyramine beta-hydroxylase