IN-VITRO MUTAGENESIS OF PH-20 HYALURONIDASE FROM HUMAN SPERM

The cDNA encoding PH-20 hyaluronidase from human sperm has been mutate d at five positions by in vitro mutagenesis. We have changed three aci dic amino acids and two arginine residues that are conserved in the se quence of mammalian PH-20 polypeptides as well as in the hyaluronidase s from bee and hornet venom. Of the former, the mutants [Gln113]PH-20 and [Gln249]PH-20 had no detectable enzymatic activity: the mutant [As n111]PH-20 had about 3% activity. The mutant [Thr252]PH-20 was also in active, while [Gly176]PH-20 had only about 1% activity. This indicates that the PH-20 hyaluronidases. like numerous enzymes that hydrolyze g lycosidic bonds, have acidic amino acids in their active site, Moreove r, for the binding of the substrate, the polyanion hyaluronan, arginin e residue appeal to be essential.