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

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp- 201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directe d mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activ ity, product progression, and kinetic parameters (K-m and V-max) were deter mined on polygalacturonic acid for the purified mutated enzymes, and bond c leavage frequencies on oligogalacturonates were calculated, Depending on th eir specific activity, the mutated endopolygalacturonases II were grouped i nto three classes, The mutant enzymes displayed bond cleavage frequencies o n penta- and/or hexagalacturonate different from the wild type endopolygala cturonase II. Eased on the biochemical characterization of endopolygalactur onase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either pr imarily substrate binding (Arg-256 and Lys-258) or maintaining the proper i onization state of a catalytic residue (His-223). The individual roles of A sp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site to pology is different from the one commonly found in inverting glycosyl hydro lases.