Kinetic and mechanistic studies with bovine testicular hyaluronidase

Bovine testicular hyaluronidase exhibits hydrolase and transglycosylase activity. To assess the magnitude of each type of reaction, the time-course of hyaluronidase catalysed hyaluronic acid degradation was followed using a sensitive and specific HPLC method. The kinetic parameters Km and Vmax were calculated for purified short chain hyaluronic acid oligomers and native hyaluronic acid based on the appearance of unreactive hyaluronic acid tetrasaccharide. For hyaluronic acid oligomers, as substrate size increased Km decreased from 2.06 to 1.09 mM while Vmax remained about the same, indicating a 5-fold increase in the enzyme-substrate association constant, k1 (kcat/Km). The values of k2 (kcat), the enzyme-substrate disassociation constant, for native hyaluronic acid and hyaluronic acid decasaccharide were similar. The value of k1 for native hyaluronic acid, however, was larger by 70-fold. Kinetic degradation mechanisms for each hyaluronic acid oligomer, using chemical-reaction kinetics, were proposed and evaluated by computer curve fitting analysis of the experimental time vs. concentration data. The derived rate constants, together with mass balance calculations, revealed that transglycosylation plays a significant role in the degradation of all hyaluronic acid oligomers studied.