Ionic interactions near the loop L4 are important for maintaining the active-site environment and the dimer stability of (pro)caspase 3.

We have examined the role of a salt bridge between Lys(242) and Glu(246) in loop L4 of procaspase 3 and of mature caspase 3, and we show that the interactions are required for stabilizing the active site. Replacing either of the residues with an alanine residue results in a complete loss of procaspase 3 activity. Although both mutants are active in the context of the mature caspase 3, the mutations result in an increase in K-m and a decrease in k(cat) when compared with the wild-type caspase 3. In addition, the mutations result in an increase in the pK(a) value associated with a change in k(cat) with pH, but does not affect the transition observed for K-m versus pH. The mutations also affect the accessibility of the active-site solvent as measured by tryptophan fluorescence emission in the presence of quenching agents and as a function of pH. We show that, as the pH is lowered, the (pro)caspase dissociates, and the mutations increase the pH-dependent instability of the dimer. Overall, the results suggest that the contacts lost in the procaspase as a result of replacing Lys(242) and Glu(246) are compensated partially in the mature caspase as a result of new contacts that are known to form on zymogen processing.