Mechanistic Studies of an Amine Oxidase Derived from d-Amino Acid Oxidase
| Autor: | Paul F. Fitzpatrick, Udayanga Wanninayake, Elizabeth E. Trimmer |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
D-Amino-Acid Oxidase Amine oxidase Stereochemistry Swine D-amino acid oxidase Flavoprotein Gene Expression Biochemistry Article Fungal Proteins 03 medical and health sciences Residue (chemistry) Glucose Oxidase Structure-Activity Relationship Catalytic Domain Phenethylamines Escherichia coli Organic chemistry Animals Enzyme kinetics Monoamine Oxidase chemistry.chemical_classification Oxidase test 030102 biochemistry & molecular biology biology Deuterium Exchange Measurement Hydrogen-Ion Concentration Amino acid Kinetics 030104 developmental biology chemistry biology.protein Biocatalysis Thermodynamics Amine gas treating Aspergillus niger |
| Zdroj: | Biochemistry. 56(14) |
| ISSN: | 1520-4995 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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