A SINGLE CALCIUM-BINDING SITE IS CRUCIAL FOR THE CALCIUM-DEPENDENT THERMAL-STABILITY OF THERMOLYSIN-LIKE PROTEASES

Thermostable thermolysin-like proteases (TLPs), such as the TLP of Bac illus stearothermophilus CU-21 (TLP-ste), bind calcium in one double ( Ca1,2) and two single (Ca3, Ca4) calcium binding sites. The single sit es are absent in thermolabile TLPs, suggesting that they are determina nts of (variation in) TLP stability. Mutations in the Ca3 and Ca4 site s of TLP-ste indeed reduced thermal stability, but only mutations in t he Ca3 site affected the calcium-dependence of stability. The predomin ant effect of the Ca3 site results from the fact that the Ca3 site is part of a region of TLP-ste, which unfolding is crucial for thermal in activation. Thermal inactivation is not caused by the absence of calci um from the Ca3 site per se, but rather by unfolding of a region of TL P-ste for which stability depends on the occupancy of the Ca3 site. In accordance with this concept is the observation that the effects of m utations in the Ca3 site could be compensated by stabilizing mutations near this site. In addition, it was observed that the contribution of calcium binding to the Ca3 was substantially reduced in extremely sta ble TLP-ste variants containing multiple stabilizing mutations in the Ca3 region. Apparently, in these latter variants, unfolding of the Ca3 region contributes little to the overall process of thermal inactivat ion.