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

Biochim Biophys Acta. 2004 Oct 1;1702(1):19-32. doi: 10.1016/j.bbapap.2004.07.005.

Abstract

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 approximately 8 is observed, which is attributed to the ionization of the alphaNH2 group of the bound substrate. Removal of S335-OH thus suppresses an apparent pK approximately 6. Both wild-type RgDAAO and the S335G mutant exhibit a substantial deuterium solvent kinetic isotope effect (> or =4) at pH<7 that disappears with increasing pH and reflects a pKapp=6.9 +/- 0.4. Interestingly, the substitution suppresses the activity towards d-lactate, suggesting a role of the serine 335 in removal of the substrate alpha-OH hydrogen.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Catalytic Domain
  • D-Amino-Acid Oxidase / chemistry*
  • D-Amino-Acid Oxidase / genetics
  • D-Amino-Acid Oxidase / metabolism*
  • DNA, Fungal / genetics
  • Hydrogen-Ion Concentration
  • Kinetics
  • Ligands
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Oxidation-Reduction
  • Rhodotorula / enzymology*
  • Rhodotorula / genetics
  • Serine / chemistry
  • Static Electricity
  • Substrate Specificity

Substances

  • DNA, Fungal
  • Ligands
  • Serine
  • D-Amino-Acid Oxidase