Role of arginine 285 in the active site of Rhodotorula gracilis D-amino acid oxidase. A site-directed mutagenesis study.
Arg(285), one of the very few conserved residues in the active site of d-amino acid oxidases, has been mutated to lysine, glutamine, aspartate, and alanine in the enzyme from the yeast Rhodotorula gracilis (RgDAAO). The mutated proteins are all catalytically competent. Mutations of Arg(285) result in an increase ( approximately 300-fold) of K(m) for the d-amino acid and in a large decrease ( approximately 500-fold) of turnover number. Stopped-flow analysis shows that the decrease in turnover is paralleled by a similar decrease in the rate of flavin reduction (k(2)), the latter still being the rate-limiting step of the reaction. In agreement with data from the protein crystal structure, loss of the guanidinium group of Arg(285) in the mutated DAAOs drastically reduces the binding of several carboxylic acids (e.g. benzoate). These results highlight the importance of this active site residue in the precise substrate orientation, a main factor in this redox reaction. Furthermore, Arg(285) DAAO mutants have spectral properties similar to those of the wild-type enzyme, but show a low degree of stabilization of the flavin semiquinone and a change in the redox properties of the free enzyme. From this, we can unexpectedly conclude that Arg(285) in the free enzyme form is involved in the stabilization of the negative charge on the N(1)-C(2)=O locus of the isoalloxazine ring of the flavin. We also suggest that the residue undergoes a conformational change in order to bind the carboxylate portion of the substrate/ligand in the complexed enzyme.
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Department of Structural and Functional Biology, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy.
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Motteran
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Vegezzi
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D017124:Conserved Sequence; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012248:Rhodotorula / Q000201:enzymology*
D017931:DNA Primers; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D001120:Arginine; D003605:D-Amino-Acid Oxidase