Kinetics of Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction.
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 compared to bovine testicular hyaluronidase (BTH), a commercially 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 electrophoresis (CZE) method. Surprisingly, the HA hexasaccharide, which is generally accepted to be the minimum substrate of BTH, was not a substrate of recombinant human PH-20 and Hyal-1. However, HA octasaccharide was converted efficiently by both enzymes, thus representing the minimum substrate for human PH-20 and Hyal-1. Additionally, BTH was shown to catabolize the HA hexasaccharide at pH 4.0 mainly by hydrolysis, while at pH 6.0 transglycosylation prevailed. Human PH-20 was found to catalyze both hydrolysis and transglycosylation of the HA octasaccharide. On the contrary, human Hyal-1 converted the HA octasaccharide mainly by hydrolysis with transglycosylation products occurring only at high substrate concentrations (> or = 500 microM). 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.
Hofinger
Edith S A
ES
Lehrstuhl für Pharmazeutische und Medizinische Chemie II, Institut für Pharmazie, Universität Regensburg, D-93040 Regensburg, Germany.
Bernhardt
Günther
G
Buschauer
Armin
A
D000818:Animals; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D004330:Drosophila; D019075:Electrophoresis, Capillary; D006031:Glycosylation; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000737:chemistry* / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D011134:Polysaccharides / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013291:Streptococcus / Q000378:metabolism; D013379:Substrate Specificity
D015815:Cell Adhesion Molecules; D011134:Polysaccharides; D006821:Hyaluronoglucosaminidase; C066852:hyaluronidase PH-20