Studies on the reaction mechanism of Rhodotorula gracilis D-amino-acid oxidase -: Role of the highly conserved Tyr-223 on substrate binding and catalysis

We have studied D-amino-acid oxidase from Rhodotorula gracilis by site-directed mutagenesis for the purpose of determining the presence or absence of residues having a possible role in acid/base catalysis, Tyr-223, one of the very few conserved residues among D-amino-acid oxidases, has been mutated to phenylalanine and to serine, Both mutants are active catalysts in turnover with D-alanine, and they are reduced by D-alanine slightly faster than wild type enzyme. The Tyr-223 -> Phe mutant is virtually identical to the wild-type enzyme, whereas the Tyr-223 -> Ser mutant exhibits 60-fold slower substrate binding and at least 800-fold slower rate of product release relative to wild-type. These data eliminate Tyr-223 as an active site acid/base catalyst. These results underline the importance of Tyr-223 for substrate binding and exemplify the importance of steric interactions in RgDAAO catalysis.