KINETIC AND MECHANISTIC STUDIES WITH BOVINE TESTICULAR HYALURONIDASE

Bovine testicular hyaluronidase exhibits hydrolase and transglycosylas e activity. To assess the magnitude of each type of reaction, the time -course of hyaluronidase catalysed hyaluronic acid degradation was fol lowed using a sensitive and specific HPLC method. The kinetic paramete rs K-max and V-max, were calculated for purified short chain hyaluroni c acid oligomers and native hyaluronic acid based on the appearance of unreactive hyaluronic acid tetrasaccharide. For hyaluronic acid oligo mers, as substrate size increased K-m decreased from 2.06 to 1.09 mM w hile V-max remained about the same, indicating a 5-fold increase in th e enzyme-substrate association constant, k(1)(k(cat)/K-m), The values of k(2) (k(cat)), the enzyme-substrate disassociation constant, for na tive hyaluronic acid and hyaluronic acid decasaccharide were similar. The value of k(1) for native hyaluronic acid, however, was larger by 7 0-fold. Kinetic degradation mechanisms for each hyaluronic acid oligom er, using chemical-reaction kinetics, were proposed and evaluated by c omputer curve fitting analysis of the experimental time vs. concentrat ion data. The derived rate constants, together with mass balance calcu lations, revealed that transglycosylation plays a significant role in the degradation of all hyaluronic acid oligomers studied.