Stabilizing mutations and calcium-dependent stability of subtilisin

Stability is a property of subtilisin which has proven particularly amenabl e to enhancement via random mutagenesis and screening, yet the effects of m ost stabilizing mutations are not understood in structural and energetic de tail. This paper seeks to explain the longstanding observation that stabili zing mutations are usually calcium-dependent in their stabilizing effect, i rrespective of their proximity to the calcium binding sites. Stabilizing mu tations in subtilisin fall into one of three classes. The largest class of mutations stabilize only in the presence of excess calcium. A smaller numbe r of mutations stabilize independently of [calcium], and a few mutations st abilize only in the presence of chelating agents, such as EDTA. This study compares the effects of mutations from each class when introduced into subt ilisin BPN ' and two calcium-free versions of subtilisin. The calcium-depen dent effects of mutations can be explained by considering subtilisin to be in conformational equilibrium between two structurally similar but energeti cally distinct states: N and N*. The equilibrium from the N* to the N state can be altered either by calcium binding to site A or by mutation. Mutatio ns which stabilize only in the presence of calcium stabilize the N state re lative to N*. Mutations which stabilize only in the presence of chelants st abilize the N* state relative to N. As a byproduct of this analysis, we hav e developed a hyperstable variant of subtilisin whose inactivation at high temperature in the presence of EDTA is 10(5) times slower than wild-type su btilisin.