Difference in substrate binding processes between intact and iodine‐inactivated lysozyme

The binding of glycol chitin to intact and iodine‐inactivated lysozyme was studied by measuring the absorbance of the complex with N ‐methylnicotinamide chloride, which binds to the subsite C in lysozyme as a competitive inhibitor. The association constant of glycol chitin to inactivated lysozyme was determined from static experiments to be 1.7 × 10 4 M −1 . The kinetics of the substrate binding to intact and iodine‐inactivated lysozyme were measured by the stopped‐flow method at 23°C and pH 5.6. The binding to inactivated lysozyme was clearly monophasic, whereas in intact lysozyme it consisted of multiple phases. In the substrate binding to intact lysozyme, a fast bimolecular process and two subsequent slow unimolecular processes were observed besides the hydrolysis process of polymer substrate. These slow phases were missing completely in inactivated lysozyme. It results from the alteration in the local structure occurring at the subsite D in inactivated lysozyme. These results mean that the slow phases are important for catalytic action of lysozyme. The rate constants of association and dissociation in the fast bimolecular process were determined in this paper. Furthermore, the association constant of the substrate to intact lysozyme was also determined kinetically to be 6.5 × 10 3 M −1 .