Limited proteolysis of thermolysin by subtilisin: isolation and characterization of a partially active enzyme derivative.

Incubation of the neutral metalloendopeptidase thermolysin at pH 9-10 in the presence of 10 mM CaCl2 for 2 days at room temperature with subtilisin at a 50:1 molar ratio leads to a derivative possessing lower (~3%) but intrinsic catalytic activity. This derivative, called thermolysin S, was isolated by gel filtration in ~ 80% yield and then separated from some residual intact thermolysin by an affinity chromatographic step on Sepharose-Gly-d-Phe. It was found that thermolysin S results from a tight association of two polypeptide fragments of apparent Mr of 24000 and 10000. Dissociation of the complex was achieved under strong denaturing conditions, such as gel filtration on a column equilibrated and eluted with 5 M guanidine hydrochloride. The positions of the clip sites were defined by amino acid analysis, end-group determination, and amino acid sequencing of the isolated fragments and shown to lie between Thr-4 and Ser-5, between Thr-224 and Gln-225, and also between Gln-225 and Asp-226. Thermolysin S, which is therefore a stable complex of fragments 5-224(225) and 225(226)-316, shows a shift in optimum pH of about 1 unit toward the acid range with respect to intact thermolysin and a Km essentially unchanged, with furylacryloyl-Gly-Leu-NH2 as substrate. Inhibitors of thermolysin such as ethoxyformic anhydride and Zn2+ ions inactivate also the nicked enzyme. The overall conformational properties of thermolysin S appear very similar to those of the parent native enzyme, as judged by circular dichroism measurements and by the fact that rabbit antiserum against native thermolysin recognizes and precipitates thermolysin S, as detected by immunodiffusion. The lower activity of thermolysin S with respect to thermolysin can be related to the fact that cleavage occurs near the Asp-226 residue interacting with His-231, involved in the catalytic function of the enzyme. Inspection of the three-dimensional structure of thermolysin obtained crystallographically reveals that subtilisin cleaves at exposed loops of the protein molecule; in addition, the sites of cleavage are characterized by highest mobility in native thermolysin [Holmes, M. A., & Matthews, B. W. (1982) J. Mol. Biol. 160, 623-639]. © 1985, American Chemical Society. All rights reserved.