THE 2 ANGSTROM 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 PMS F-inhibited subtilisin E was solved by molecular replacement followed by refinement with the X-PLOR program. This resulted in the 2.0 Angstr om structure of subtilisin E with an R-factor of 0.191 for 8-2 Angstro m data and r.m.s. deviations from ideal values of 0.021 Angstrom and 2 .294 degrees for bond lengths and bond angles respectively. The PMSF g roup 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 s ubtilisins. The calcium-binding sites are different in detail in the t wo 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 molecul e in the mutant structure, which is hydrogen bonded to side chains of Ser118 and its neighbouring residues Lys27 and Asp120.