Properties and chemical modification of D-amino acid oxidase from Trigonopsis variabilis |
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Authors: | Thomas Schräder Jan R Andreesen |
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Institution: | Institut für Mikrobiologie, Universit?t Halle, Weinbergweg 16 a, D-06099 Halle, Germany Tel. +49-345-5526350; Fax +49-345-5527010 e-mail: J.Andreesen@mikrobiologie.uni-halle.de, DE
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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.
Received: 22 August 1995 / Accepted: 17 October 1995 |
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Keywords: | d-amino acid oxidase Trigonopsis variabilis Chemical modification Histidine residues Km Vmax Sulfite |
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