The role of bound calcium ions in thermostable, proteolytic enzymes. II. Studies on thermolysin, the thermostable protease from Bacillus thermoproteolyticus

Abstract

The functional properties of the four calcium ions, bound by thermolysin, appear to be very similar to those of the single calcium ion bound by thermomycolase (G. Voordouw and R.S. Roche (1975), Biochemistry, preceding paper in this issue). Hence when the free calcium ion concentration is varied in the range where the calcium double-site dissociates (G. Voordouw and R.S. Roche (1974), Biochemistry 13, 5017), no changes are observed in the sedimentation coefficient or the peptide circular dichroism. Differences in molar ellipticity and molar extinction coefficient occur in the aromatic ultraviolet region, which parallel the occupancy of the calcium binding double site. The difference spectrum, characterized by a main band at 290 nm and a somewhat smaller band at 283 nm, is interpreted as due to the transfer of a partially buried tryptophan residue to the aqueous solvent upon dissociation of the two calcium ions from the double site. This is most likely Trp-186, which is in between Asp-185 and Glu-187, two chelating amino acids of this site. From the calcium dependence of the rate constant for autolytic degradation we conclude, as for thermomycolase, that only conformers devoid of bound calcium ion serve as substrates in the reaction. This rate constant increases about 1000-fold, when the double site dissociates. Hydrogen-tritium exchange studies show the presence of a large stable strcutural core, comprising about 32% of all the peptide hydrogens present. These do not exchange-in after 24 hr at 25degreesC, pH 9.0, ionic strenth 0.1. The exchange-out of 60 slow hydrogens was found to be independent of the free calcium ion concentration in the range 2.0-8.0 X 10(-4) M, where all four calcium-binding sites are saturated. The calcium dependence of the first-order rate constant for thermal denaturation at 80degreesC, pH 7.0, indicates that thermolysin is stabilized by only one calcium ion under these conditions. These observations are rationalized in terms of a calcium-binding model for thermolysin and the known three-dimensional structure of the enzyme and its calcium-binding sites.