Open AccessCrystallographic Studies of Savinase, a Subtilisin‐like Proteinase, at pH 10.5
Author(s) -
Lange Gudrun,
Betzel Christian,
Branner Sven,
Wilson Keith S.
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.00507.x
Subject(s) - subtilisin , histidine , chemistry , active site , protonation , serine , stereochemistry , binding site , substrate (aquarium) , serine protease , crystallography , enzyme , biochemistry , biology , protease , organic chemistry , ion , ecology
The effect of high pH (pH 10.5) on Savinase, a subtilisin‐like serine proteinase, has been investigated using X‐ray crystallography. The structures of two Savinase mutants were determined at two different pH values, namely pH 6.0, where the enzyme is inactive (this is the pH at which most of the structural work has been carried out on other serine proteinases), and pH 10.5, where Savinase is active. Comparison of these high resolution (0.16 nm) structures showed four related sets of changes between the two pH values. First, the difference in protonation state of the active‐site histidine leads to a change in conformation of the active‐site triad. Secondly, there are resulting changes in the water structure around this histidine residue with much increased mobility of the water in the active‐site at pH 10.5. Thirdly, the two substrate‐binding loops on either side of the binding site are less well linked by ordered water molecules at pH 10.5 and show substantially increased flexibility. Finally, the first substrate‐binding loop, residues at positions 99–104, moves slightly so as to widen the substrate channel.