On the mechanism of Rhodotorula gracilis D-amino acid oxidase : role of the active site serine 335

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2004
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Boselli, Angelo
Piubelli, Luciano
Molla, Gianluca
Sacchi, Silvia
Pilone, Mirella S.
Pollegioni, Loredano
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Serine 335 at the active site of D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) is not conserved in other DAAO sequences. To assess its role in catalysis, it was mutated to Gly, the residue present in mammalian DAAO, an enzyme with a 35-fold lower turnover number with D-alanine. The spectral and ligand binding properties of the S335G mutant are similar to those of wild-type enzyme, suggesting an active site with minimally altered electrostatic properties. The S335G mutant is catalytically active, excluding an essential role of S335 in catalysis. However, S335-OH contributes to the high efficiency of the mutant enzyme since the catalytic activity of the latter is lower due to a decreased rate of flavin reduction relative to wild-type RgDAAO. Catalytic rates are pH-dependent and appear to converge to very low, but finite and similar values at low pH for both wild-type and S335G RgDAAO. While this dependence exhibits two apparent pKs with wild-type RgDAAO, with the S335G mutant a single, apparent pK ≈8 is observed, which is attributed to the ionization of the αNH2 group of the bound substrate. Removal of S335-OH thus suppresses an apparent pK≈6. Both wild-type RgDAAO and the S335G mutant exhibit a substantial deuterium solvent kinetic isotope effect (≥4) at pH

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570 Biowissenschaften, Biologie
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D-Amino acid oxidase, Flavoprotein, Site-directed mutagenesis, Reaction mechanism, pH effect, Proton inventory
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ISO 690BOSELLI, Angelo, Luciano PIUBELLI, Gianluca MOLLA, Silvia SACCHI, Mirella S. PILONE, Sandro GHISLA, Loredano POLLEGIONI, 2004. On the mechanism of Rhodotorula gracilis D-amino acid oxidase : role of the active site serine 335. In: Biochimica et Biophysica Acta / Proteins and Proteomics. 2004, 1702(1), pp. 19-32. ISSN 1570-9639. Available under: doi: 10.1016/j.bbapap.2004.07.005
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@article{Boselli2004mecha-6817,
  year={2004},
  doi={10.1016/j.bbapap.2004.07.005},
  title={On the mechanism of Rhodotorula gracilis D-amino acid oxidase : role of the active site serine 335},
  number={1},
  volume={1702},
  issn={1570-9639},
  journal={Biochimica et Biophysica Acta / Proteins and Proteomics},
  pages={19--32},
  author={Boselli, Angelo and Piubelli, Luciano and Molla, Gianluca and Sacchi, Silvia and Pilone, Mirella S. and Ghisla, Sandro and Pollegioni, Loredano}
}
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