Effects on subtilisin BPN

A. A. Kossiakoff, M. Ultsch, S. White, C. Eigenbrot, Neutron Structure of Subtilisin BPN Effects of Chemical Environment on Hydrogen-Bonding Geometries and the Pattern of Hydrogen-Deuterium Exchange in Secondary Structure Elements , Biochemistry 1991, 30, 1211-1221. 

Support for an even more remote electrostatic effect on the mechanism is supplied by the studies of Jackson and Fersht (1993). They mutated charged residues on the surface of Subtilisin BPN, that are 13-15A from the active site, to either neutral or oppositely charged residues. The effect of those mutations on the inhibition constant, Ki, of a trifluoromethyl ketone, was compared for wt and mutated subtilisin. The mutations were Asp-36 (located on a surface loop outside the active site cleft and separated from His-64 by about 15-16A) to Gin, and of Asp-99 (about 12-13A from His-64) to Ser and to Lys. The active site of subtilisin includes Ser-221, His-64 and Asp-32. 

---