title: In vitro mutagenesis of PH-20 hyaluronidase from human sperm.
abstract: The cDNA encoding PH-20 hyaluronidase from human sperm has been mutated at five positions by in vitro mutagenesis. We have changed three acidic amino acids and two arginine residues that are conserved in the sequence of mammalian PH-20 polypeptides as well as in the hyaluronidases from bee and hornet venom. Of the former, the mutants [Gln113]PH-20 and [Gln249]PH-20 had no detectable enzymatic activity; the mutant [Asn111]PH-20 had about 3% activity. The mutant [Thr252]PH-20 was also inactive, while [Gly176]PH-20 had only about 1% activity. This indicates that the PH-20 hyaluronidases, like numerous enzymes that hydrolyze glycosidic bonds, have acidic amino acids in their active site. Moreover, for the binding of the substrate, the polyanion hyaluronan, arginine residues appear to be essential.
authors.affiliation: Institut für Molekularbiologie, Osterreichische Akademie der Wissenschaften, Salzburg, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Arming
authors.forename: B
authors.initials: B
authors.lastname: Strobl
authors.forename: C
authors.initials: C
authors.lastname: Wechselberger
authors.forename: G
authors.initials: G
authors.lastname: Kreil
mesh_terms: D000595:Amino Acid Sequence; D018076:DNA, Complementary; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics; D013094:Spermatozoa / Q000201:enzymology*; D014616:Vaccinia virus / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D006821:Hyaluronoglucosaminidase