[Thermitase--a thermostable serine protease. VI. Kinetic and fluorescence studies on the interaction of the enzymes with dansylated peptide chloromethylketones].

The modification of thermitase with dansylated peptide chloromethyl ketones Dns-Alan-PheCH2Cl (n = 1 to 4) was investigated by means of kinetic and fluorescence methods. Furthermore the influence of dimethyl formamide and dimethyl sulfoxide on the enzyme activity is reported. The synthesis of the Dns-substituted chloromethyl ketones acting as irreversible inhibitors was started from the analogous Z-compounds. The substances Dns-Alan-PheCH2-Cl with n = 1, 2, and 3 have lower inhibition effects than the sequence homologous Z-protected peptide chloromethyl ketones. The fluorescence measurements showed that the dansyl group forms hydrophobic interactions with the enzyme and takes part in energy transfer processes particularly in the case of thermitase labelled with Dns-Ala2-PheCH2Cl. From the kinetic data of the Dns- and Z-protected inhibitors it is concluded that thermitase presents five subsites at the S-site of its active center. This confirms our earlier assumption that the topology of the active center of thermitase is comparable with that of subtilisin BPN'.