Preparation and characterization of a site-directed mutant of Escherichia coli L-threonine dehydrogenase

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Title: Preparation and characterization of a site-directed mutant of Escherichia coli L-threonine dehydrogenase
Author: Zhou, Qian
Abstract: Threonine dehydrogenase from E.coli is a member of the medium-chain, Zn2+-containing alcohol dehydrogenase family. A site-directed mutant of theronine dehydrogenase was prepared at position 88 by replacing the native glutamate with cysteine using polymerase chain reaction. The mutated enzyme, designated TDH E88C, was expressed and purified to homogeneity in a three-step chromatographic procedure. The activity of the mutant after each step of purification was determined by a specific assay. Both the total activity and the specific activity of TDH E99C were significantly decreased compared to the wild-type TDH. The mutant enzyme was not sufficiently stable to obtain reliable Vmax or Km values, preventing a quantitative comparison to the wild type enzyme. Therefore, a definitive conclusion on the role of the Glu-88 remains unresolved, pending further studies in the future.
Record URI: http://hdl.handle.net/1850/13945
Date: 1999-11

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