Mechanistic Studies of an Amine Oxidase Derived from d -Amino Acid Oxidase

Elizabeth E. Trimmer, Udayanga S. Wanninayake, Paul F Fitzpatrick

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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/Km value for the amine, consistent with the neutral amine being the substrate. The deuterium isotope effect on the kcat/Km value 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/KO2 value 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.

Original languageEnglish (US)
Pages (from-to)2024-2030
Number of pages7
JournalBiochemistry
Volume56
Issue number14
DOIs
StatePublished - Apr 11 2017

ASJC Scopus subject areas

  • Biochemistry

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    Trimmer, E. E., Wanninayake, U. S., & Fitzpatrick, P. F. (2017). Mechanistic Studies of an Amine Oxidase Derived from d -Amino Acid Oxidase. Biochemistry, 56(14), 2024-2030. https://doi.org/10.1021/acs.biochem.7b00161