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...

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Bibliographic Details
Published in:The Journal of biological chemistry 1999-12, Vol.274 (51), p.36233-36240
Main Authors: Harris, C M, Molla, G, Pilone, M S, Pollegioni, L
Format: Article
Language:English
Subjects:
Binding Sites - genetics
Catalysis
Conserved Sequence
D-Amino-Acid Oxidase - chemistry
D-Amino-Acid Oxidase - genetics
D-Amino-Acid Oxidase - metabolism
Kinetics
Mutagenesis, Site-Directed
Rhodotorula
Rhodotorula gracilis
Structure-Activity Relationship
Substrate Specificity
Tyrosine
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Summary: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.
ISSN:0021-9258
DOI:10.1074/jbc.274.51.36233