Role of Calcium in the thermal stability of thermolysin

The effect of calcium ion on the thermal stability of thermolysin has been investigated. The native protein undergoes an irreversible structural change and autolysis at high temperature. Analysis of the calcium ion dependence of the apparent melting temperature observed spectroscopically gives an apparent deltaH of -x (130 kcal/mol) where x is either 1 or 2. Neither zinc ion, where bound at the active site, nor terbium ion, which binds very tightly to the double calcium binding site, shows a stabilizing effect. These sites are therefore presumably not coupled to the transition which leads to autolysis. Removal of calcium ion from the native enzyme at temperatures below 50 degrees C results in inactivation but not major autolysis. The addition of 1 equiv of terbium before calcium removal results in a protein species which is 40% active and is no longer subject to thermal stabilization by calcium. These results suggest a pathway for the thermal inactivation of the enzyme which involves an irreversible structural change at one or both of the single calcium ion sites. This change propagates to the active site and results in inactivation. The binding of calcium ion to either or both single sites completely inhibits this structural change. The structural change is apparently cooperative and may correspond to a localized denaturation of the native structure.