Fluorescence labeled and cross-linked subtilisin: kinetic characteristics and binding to Streptomyces subtilisin inhibitor

In the preceding paper, the preparation of fluorescent cross-linked subtilisin was described. In this paper we present the catalytic and binding properties of the modified enzyme. Kinetic analysis showed that the cross-linked dimeric subtilisin retained both catalytic activity and binding affinity toward synthetic substrates. These kinetic characteristics of the modified enzyme were nearly identical to those of the native enzyme. The modified enzyme also exhibited a specific interaction with Streptomyces subtilisin inhibitor (SSI) with 1 to 1 stoichiometry. The formation of a polymeric complex, which is the expected product with cross-linked subtilisin, was demonstrated.