title: Abnormally high pKa of an active-site glutamic acid residue in Bacillus circulans xylanase. The role of electrostatic interactions.
abstract: The active site of Bacillus circulans xylanase (1,4-beta-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 pKa 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.
authors.affiliation: Department of Biochemistry, University of Ottawa, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Davoodi
authors.forename: W W
authors.initials: WW
authors.lastname: Wakarchuk
authors.forename: R L
authors.initials: RL
authors.lastname: Campbell
authors.forename: P R
authors.initials: PR
authors.lastname: Carey
authors.forename: W K
authors.initials: WK
authors.lastname: Surewicz
mesh_terms: D000838:Anions; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D043364:Endo-1,4-beta Xylanases; D004795:Enzyme Stability; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D017186:Titrimetry; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000838:Anions; D011522:Protons; D018698:Glutamic Acid; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases