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.
Harris
C M
CM
Department of Structural and Functional Biology, University of Insubria, via J. H. Dunant 3, 21100 Varese, Italy.
Molla
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G
Pilone
M S
MS
Pollegioni
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D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D012248:Rhodotorula; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine
D014443:Tyrosine; D003605:D-Amino-Acid Oxidase