The active-site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis

S. Armand, M.J.M. Wagemaker, P. Sanchez-Torres, H.C.M. Kester, Y. van Santen, B.W. Dijkstra, J. Visser, J.A.E. Benen

    Research output: Contribution to journalArticleAcademicpeer-review

    79 Citations (Scopus)

    Abstract

    Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (Km and Vmax) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.
    Original languageEnglish
    Pages (from-to)691-696
    JournalJournal of Biological Chemistry
    Volume275
    DOIs
    Publication statusPublished - 2000

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