The effect of chlorambucil upon lysozyme activity

Chicken egg white lysozyme is progressively inhibited by diazoacetyl-DL-nor reucine methyl ester (DANME) and by chlorambucil at concentrations of 3.4 x 10(-3) M and 5 x 10(-3) M respectively over a three-hour time period. DANM E inhibits lysozyme activity to the extent of 87%, and chlorambucil inhibit s the enzyme to the extent of 93%. N,N',N "-triacetylchitotriose [(NAG)(3)] , which binds to subsites A, B and C of the enzyme protects lysozyme from D ANME inhibition to the extent of 40% of the total activity when added to th e enzyme at a concentration of 3.6 x 10(-3) M prior to the addition of DANM E. (NAG)(3) protects the enzyme from inhibition by chlorambucil to the exte nt of 14% of the total activity when added to the enzyme at a concentration of 5.6 x 10(-3) M prior to the addition of chlorambucil. Since DANME react s exclusively with carboxyl groups, and since aspartic acid 101 is required for binding the carbohydrate substrate at site A, it is suggested that (NA G)(3) may bind reversibly to the active site of the enzyme, thereby protect ing aspartic acid 101 from esterification by DANME and subsequent inactivat ion. Chlorambucil, which may react with carboxyl, amino, imidazole and thio l groups, more likely acts upon a larger number of susceptible sites, there by causing irreversible alkylation and conformation changes. As a bifunctio nal alkylating agent, it may also cross-link with two available nucleophile s. The K-m for lysozyme with M. lysodeikticus as a substrate in wholly aque ous medium was determined to be 0.05 mg/mL. The inhibitor exhibits a partia lly uncompetitive inhibition upon pre-incubation with the enzyme, and a mix ed inhibition between competititve and noncompetitive when pre-incubated wi th the substrate. (C) Elsevier, Paris.