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Savinase and Esperase (EC. 3.4.21.14) are secreted by the alkalophilic bacterium Bacillus lentus and are representatives of that subgroup of subtilisin enzymes with maximum stability in the range of pH 7 to 10 and high activity in the range of pH 8 to 12. The crystal structures of native Savinase and diisopropyl fluorophosphate (DFP) inhibited Esperase have been refined using X-ray data to 1.4 angstroms and 1.8 angstroms resolution respectively collected with synchrotron radiation. The structures were refined to R-factors (=(Sigma//Fo/-/Fc//)/(Sigma/Fo/)) of 16.4% for Esperase and 17.3% for Savinase. The sequence identity between the two enzymes is 66%. The structures are very similar to those of other Bacillus subtilisins. There are two calcium ions in each, equivalent to the strong and the weak sites in subtilisins Carlsberg and BPN'. The structures show novel features which can to some extent be related to their stability and activity. The large number of salt bridges in Esperase and Savinase is likely to contribute to the high thermal stability. Non-conservative substitutions and deletions in the hydrophobic binding pocket S1 as well as the more hydrophobic character of the substrate binding region probably contribute to the alkaline activity profile of the enzymes. Towards the end of the binding site there is an extra proline, Pro131, in Savinase near proline 129, forming a cluster that provides extra active-site rigidity compared with other subtilisins. On the other side of the active site of Esperase and Savinase, the tyrosine found in most other subtilisins is replaced by leucine and valine respectively. The tyrosine potentially interacts with substrate residue P6. At high pH, the negatively charged deprotonated tyrosine could interact unfavorably with the substrate, a possibility that is overcome by substitution with a neutral residue. This is probably one explanation for the shift of the activity profile of Esperase and Savinase to more alkaline pH.

Savinase (EC3.4.21.14) is secreted by the alkalophilic bacterium Bacillus lentus and is a representative of that subgroup of subtilisin enzymes with maximum stability in the pH range 7 to 10 and high activity in the range 8 to 12. It is therefore of major industrial importance for use in detergents. The crystal structure of the native form of Savinase has been refined using X-ray diffraction data to 1.4 A resolution. The starting model was that of subtilisin Carlsberg. A comparison to the structures of the closely related subtilisins Carlsberg and BPN' and to the more distant thermitase and proteinase K is presented. The structure of Savinase is very similar to those of homologous Bacillus subtilisins. There are two calcium ions in the structure, equivalent to the strong and the weak calcium-binding sites in subtilisin Carlsberg and subtilisin BPN', well known for their stabilizing effect on the subtilisins. The structure of Savinase shows novel features that can be related to its stability and activity. The relatively high number of salt bridges in Savinase is likely to contribute to its high thermal stability. The non-conservative substitutions and deletions in the hydrophobic binding pocket S1 result in the most significant structural differences from the other subtilisins. The different composition of the S1 binding loop as well as the more hydrophobic character of the substrate-binding region probably contribute to the alkaline activity profile of the enzyme. The model of Savinase contains 1880 protein atoms, 159 water molecules and two calcium ions. The crystallographic R-factor [formula; see text].