Open AccessAbnormally High pK a of an Active‐Site Glutamic Acid Residue in Bacillus Circulans Xylanase
Author(s) -
Davoodi Jamshid,
Wakarchuk Warren W.,
Campbell Robert L.,
Carey Paul R.,
Surewicz Witold K.
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.0839a.x
Subject(s) - bacillus circulans , xylanase , chemistry , active site , titration , residue (chemistry) , enzyme , carboxylate , glutamic acid , stereochemistry , biochemistry , amino acid , inorganic chemistry
The active site of Bacillus circulans xylanase (1,4‐β‐ D ‐xylanohydrolase, EC 3.2.1.8) contains two glutamic acid residues, Glu78 and Glu172, which are crucial for the catalytic activity of the enzyme. Fourier‐transform infrared spectroscopy was used to determine the ionization state of these residues as a function of pH. For the wild‐type enzyme, titration of one of the carboxylate groups occurs at pH 6.8. This titration is absent in the Glu78→Gln and Glu172→Gln variants of the enzyme. This, together with crystallographic data, indicates that Glu172 has an abnormally high pK a of 6.8, caused largely by electrostatic interactions of this residue with the proximal Glu78. Differential scanning calorimetry experiments with the wild‐type xylanase and a number of its mutants have shown that the presence of two nearby carboxyl groups results in a pH‐dependent destabilization of the protein structure.