Action of Chondroitinases<subtitle>III. Ionic Strength Effects and Kinetics in the Action of Chondroitinase AC</subtitle>

Abstract

In order to compare the action of Arthrobacter chondroitinase AC [EC 4.2.2.51 (A-Chase) with that of Flavobacterium chondroitinase AC (F-Chase), steady-state kinetic studies were carried out at pH 6.0. Preliminary experiments showed the action of A-Chase to be accelerated by the addition of a salt to the reaction mixture. The degree of activation by the salt additive was maximum at the ionic strength μ=0.05. The action of A-Chase was suppressed by the salt at ionic strength higher than 0.05. Several bivalent metal cations such as Ca2+ Co2+ Mg2+ and Ba2+ were more effective for activation than univalent metal cations such as Na+, K+ etc. Further, A-Chase was inactivated by Pb2+, Cu2+, Fe2+ and Sn2+. Kinetic studies on the effect of ionic strength showed that an ionic additive causes both activation of the substrate and inhibition of A-Chase. It was also shown that DS is a competitive inhibitor of A-Chase; the-dissociation constant of the enzyme-inhibitor complex, K1 is 6.1 × 10−5 M as moles of repeating disaccharide unit. Heparin is not an inhibitor of the enzyme. Kinetic parameters of the action of A-Chase and F-Chase were estimated for CbS-A, CbS-C, Ch, and HA. Van't Hoff plots of the apparent Km values and Arrhenius plots of the apparent k0 values for both enzymes with these substrates gave linear relationships with the exception of the plots of k0 for F-Chase. The thermodynamic and activation parameters under 30°C were calculated assuming a Michaelis-Menten type mechanism with the enzyme and the substrate in rapid equilibrium. The ΔH values obtained for various substrates were negative for A-Chase and positive for F-Chase. The results obtained in this study suggest that the driving force of enzyme-substrate complex formation can be attributed to ionic interaction for A-Chase and to hydrophobic bonding for F-Chase.