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Archives of Microbiology

, Volume 165, Issue 1, pp 41–47 | Cite as

Properties and chemical modification of D-amino acid oxidase from Trigonopsis variabilis

  • Thomas Schräder
  • Jan R. Andreesen
Original paper

Abstract

The basic properties of purified d-amino acid oxidase from the yeast Trigonopsis variabilis were investigated. The pH optimum of activity was between pH 8.5 and 9.0, and the native molecular masses of holo- and apo-enzyme were determined to be 170 kDa; higher aggregates corresponded to molecular masses of 320 and 570 kDa. The apparent V max and K m values for different substrates varied between 3.7 to 185 U/mg and 0.2 to 17.3 mM, respectively. The reaction of d-amino acid oxidase with sulfite was followed by the typical spectral modifications of the FAD resembling the reduced enzyme; a K d of 30 μM was calculated for the N(5)-adduct. The red anionic flavin radical of the enzyme was stable; benzoate had no influence on the spectral properties. A complete loss of enzyme activity was observed after chemical modification by the histidine-specific reagent diethyl pyrocarbonate. The inactivation showed pseudo-first-order kinetics, with a second-order rate constant of 13.6 M–1 min–1 at pH 6.0 and 20°C. The addition of a substrate under anoxic conditions led to a substantial protection from inactivation, which indicates a localization of the modified residues close to the active site. The pKa of the reacting group was determined to be 7.7, and the rate of inactivation reached a limiting value of 0.031 min–1.

Key wordsd-amino acid oxidase Trigonopsis variabilis Chemical modification Histidine residues Km Vmax Sulfite 

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Copyright information

© Springer-Verlag Berlin Heidelberg 1996

Authors and Affiliations

  • Thomas Schräder
    • 1
  • Jan R. Andreesen
    • 1
  1. 1.Institut für Mikrobiologie, Universität Halle, Weinbergweg 16 a, D-06099 Halle, Germany Tel. +49-345-5526350; Fax +49-345-5527010 e-mail: J.Andreesen@mikrobiologie.uni-halle.deDE

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