Identification and role of ionizing functional groups at the active center of Rhodotorula gracilis D-amino acids oxidase

D-Amino acid oxidase (DAAO) is a flavoprotein oxidase that catalyzes the oxidation of amino acids and produces ketoacids and H2O2. The rate of product release from reduced DAAO from Rhodotorula gracilis is pH dependent and reflects a pKa of ∼9.3. Binding of benzoate and 3,3,3-trifluoro-D-alanine to wild-type and Y238F-DAAO is also pH dependent (pKa = 9.8 ± 0.1 and 9.05 ± 0.1, respectively for benzoate binding). However, binding of benzoate to Y223F-DAAO is pH independent, indicating the pKa is due to Y223-OH. This latter residue is thus involved in substrate binding, and probably is the group that governs product release. In contrast to this, the second active site tyrosine, Y238, has little influence on ligand binding. © 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.