Kinetics of Hyal-1 and PH20 hyaluronidases: Comparison of minimal substrates and analysis of the transglycosylation reaction. Glycobiology

The availability of recombinant expression systems for the production of purified human hyaluronidases PH-20 and Hyal-1 facilitated the first detailed analysis of the enzymatic reaction products. The human recombinant enzymes, both expressed by Drosophila Schneider-2 (DS-2) cells, were com-pared to bovine testicular hyaluronidase (BTH), a commer-cially available hyaluronidase preparation, which has long been considered a prototype of mammalian hyaluronidases. The conversion of low molecular weight hyaluronic acid (HA) fragments was detected by a capillary zone elec-trophoresis (CZE) method. Surprisingly, the HA hexasac-charide, which is generally accepted to be the minimum substrate of BTH, was not a substrate of recombinant hu-man PH-20 and Hyal-1. However, HA octasaccharide was converted efficiently by both enzymes, thus representing the minimum substrate for human PH-20 andHyal-1. Addition-ally, BTH was shown to catabolize the HA hexasaccharide at pH 4.0 mainly by hydrolysis, while at pH 6.0 transgly-cosylation prevailed. Human PH-20 was found to catalyze both hydrolysis and transglycosylation of the HA octasac-charide. On the contrary, human Hyal-1 converted the HA octasaccharide mainly by hydrolysis with transglycosyla-tion products occurring only at high substrate concentra-tions (≥500µM).The differences between the hyaluronidase subtypes and isoenzymes were much more prominent than expected. Obviously, the different hyaluronidase subtypes have evolved into very specialized enzymes with respect to their catalytic mechanism of action