Incubation of the neutral
metalloendopeptidase thermolysin at pH 7.2 in the presence of
EDTA and/or low concentrations of
calcium ions produces fast
enzyme inactivation as a result of
autolysis. The 'nicked'
protein is a folded species composed of three tightly associated
protein fragments. Dissociation of this complex can be achieved under denaturing conditions, such as gel filtration on a column equilibrated with 5 M
guanidine hydrochloride or reverse-phase high-performance liquid chromatography (HPLC) at acidic pH. The positions of the
peptide bond cleavages were defined by isolation of the individual fragments by HPLC and their characterization by
amino acid analysis after
acid hydrolysis, end-group determination and partial
amino acid sequencing. The results of these analyses indicated that the nicked
protein is composed of fragments 1-196, 197-204 and 205-316 and thus that the corresponding sites of limited proteolysis occur at the
polypeptide chain loop involved in the binding of Ca(4) in native
thermolysin [Matthews, B. W., Weaver, L. H. and Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The overall conformational properties of nicked
thermolysin are quite similar to those of the intact
protein, as judged by spectroscopic measurements and by the fact that rabbit
antibodies against native
thermolysin recognize and precipitate the nicked
protein in immunodiffusion assays. The nicked
protein was much less stable to heat and unfolding agents than intact
thermolysin. These results contribute to a better knowledge of the molecular mechanism of stabilization of native
thermolysin by the four bound
calcium ions and demonstrate that the function of Ca(4) is to stabilize the loop 190-205 on the surface of the molecule against
autolysis.