The 2 A crystal structure of subtilisin E with PMSF inhibitor

Using enzyme prepared by the DNA recombination technique, subtilisin E from Bacillus subtilis was crystallized in space group P2(1)2(1)2(1) with two molecules in an asymmetric unit. The crystal structure of PMSF-inhibited subtilisin E was solved by molecular replacement followed by refinement with the X-PLOR program. This resulted in the 2.0 A structure of subtilisin E with an R-factor of 0.191 for 8-2 A data and r.m.s. deviations from ideal values of 0.021 A and 2.294 for bond lengths and bond angles respectively. The PMSF group covalently bound to Ser221 appeared very clearly in the electron density map. Except for the active site disturbed by PMSF binding, the structural features of subtilisin E are almost the same as in other subtilisins. The calcium-binding sites are different in detail in the two independent molecules of subtilisin E. Based on the structure, the remarkably enhanced heat stability of mutant N118S of subtilisin E is discussed. It is very likely that there is an additional water molecule in the mutant structure, which is hydrogen bonded to side chains of Ser118 and its neighbouring residues Lys27 and Asp120.