Characterization of Enzyme-substrate Complex of Lysozyme*

Lysozyme [EC 3.2.1.17] was adsorbed on a column of insoluble substrate, carboxymethyl chitin (CM-chitin)***, by forming an enzyme-substrate complex. Column chromatography on CM-chitin showed that Trp-62 was indispensable for the binding of lysozyme to CM-chitin, while Trp-108 was not. Native or acetylated lysozyme or lysozyme oxidized with iodine was strongly adsorbed on a column of CM-chitin at pH6–10. However, lysozyme was not adsorbed on the column at pH2–5, the range where it has a hydrolytic action on the substrate, the polysaccharide of N-acetylglucosamine. This clearly indicates that there are two types of complex, nonproductive complex and productive complex. The pH-dependence of the adsorption of lysozyme showed that some binding groups in the lysozyme molecule were greatly affected by protonation of a carboxyl group with pK5.6 and that these groups were responsible for the interconversion of two types of complex.