title: Mechanistic Studies of an Amine Oxidase Derived from d-Amino Acid Oxidase.
abstract: The flavoprotein d-amino acid oxidase has long served as a paradigm for understanding the mechanism of oxidation of amino acids by flavoproteins. Recently, a mutant d-amino acid oxidase (Y228L/R283G) that catalyzed the oxidation of amines rather than amino acids was described [Yasukawa, K., et al. (2014) Angew. Chem., Int. Ed. 53, 4428-4431]. We describe here the use of pH and kinetic isotope effects with (R)-α-methylbenzylamine as a substrate to determine whether the mutant enzyme utilizes the same catalytic mechanism as the wild-type enzyme. The effects of pH on the steady-state and rapid-reaction kinetics establish that the neutral amine is the substrate, while an active-site residue, likely Tyr224, must be uncharged for productive binding. There is no solvent isotope effect on the kcat/Kmvalue for the amine, consistent with the neutral amine being the substrate. The deuterium isotope effect on the kcat/Kmvalue is pH-independent, with an average value of 5.3, similar to values found with amino acids as substrates for the wild-type enzyme and establishing that there is no commitment to catalysis with this substrate. The kcat/KO2value is similar to that seen with amino acids as the substrate, consistent with the oxidative half-reaction being unperturbed by the mutation and with flavin oxidation preceding product release. All of the data are consistent with the mutant enzyme utilizing the same mechanism as the wild-type enzyme, transfer of hydride from the neutral amine to the flavin.
authors.affiliation: Department of Chemistry, Grinnell College , Grinnell, Iowa 50112, United States.
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Trimmer
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center , San Antonio, Texas 78229, United States.
authors.forename: Udayanga S
authors.initials: US
authors.lastname: Wanninayake
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center , San Antonio, Texas 78229, United States.
authors.forename: Paul F
authors.identifier: 0000-0002-8486-2024
authors.initials: PF
authors.lastname: Fitzpatrick
mesh_terms: D000818:Animals; D001234:Aspergillus niger / Q000737:chemistry / Q000201:enzymology; D055162:Biocatalysis; D020134:Catalytic Domain; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005949:Glucose Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008995:Monoamine Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010627:Phenethylamines / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013552:Swine; D013816:Thermodynamics
chemical_list: D005656:Fungal Proteins; D010627:Phenethylamines; D005949:Glucose Oxidase; D003605:D-Amino-Acid Oxidase; D008995:Monoamine Oxidase; C033198:1-phenethylamine