Characterization of The Active Site of Schwanniomyces Occidentalis Glucoamylase by In Vitro Mutagenesis

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Abstract
Site-directed mutagenesis was performed to define the active site of the Schwanniomyces occidentalis glucoamylase. The mutated GAM1 genes were expressed in Saccharomyces cerevisiae, and enzymatic and growth properties of the transformants were determined. Mutants were transcribed and translated similar to the wild-type glucoamylase. Therefore, all effects on enzymatic activity could be referred to single amino acid substitutions. Asp470 was shown to be essential for the enzyme activity. Replacement of Asp470 by glycine led to a complete loss of activity. We suppose that Asp470 serves as a general acid-base and stabilizes the formation of the intermediate carbenium ion. Substitution of Trp468 by alanine affected predominantly the alpha-1,6 activity and not the alpha-1,4 activity of the enzyme. The exchange impaired substrate binding as well as enzymatic catalysis. An influence of amino acid 474 on the substrate specificity could not be demonstrated. Exchanges at position 474 exhibited K(m) and Vmax values similar to wild-type glucoamylase.

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