| Autor: |
Trimmer EE; Department of Chemistry, Grinnell College , Grinnell, Iowa 50112, United States., Wanninayake US; Department of Biochemistry and Structural Biology, University of Texas Health Science Center , San Antonio, Texas 78229, United States., Fitzpatrick PF; Department of Biochemistry and Structural Biology, University of Texas Health Science Center , San Antonio, Texas 78229, United States. |
| Jazyk: |
angličtina |
| Zdroj: |
Biochemistry [Biochemistry] 2017 Apr 11; Vol. 56 (14), pp. 2024-2030. Date of Electronic Publication: 2017 Apr 03. |
| DOI: |
10.1021/acs.biochem.7b00161 |
| Abstrakt: |
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 k cat /K m value for the amine, consistent with the neutral amine being the substrate. The deuterium isotope effect on the k cat /K m 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 k cat /K O 2 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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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