title: Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations.
abstract: NMR-monitored pH titration curves of proteins provide a rich source of structural and electrostatic information. Although relatively straightforward to measure, interpreting pH-dependent chemical shift changes to obtain site-specific acid dissociation constants (pK                         (A) values) is challenging. In order to analyze the biphasic titrations exhibited by the side chain (13)C(γ) nuclei of the nucleophilic Glu78 and general acid/base Glu172 in Bacillus circulans xylanase, we have revisited the formalism for the ionization equilibria of two coupled acidic residues. In general, fitting NMR-monitored pH titration curves for such a system will only yield the two macroscopic pK                         (A) values that reflect the combined effects of both deprotonation reactions. However, through the use of mutations complemented with ionic strength-dependent measurements, we are able to extract the four microscopic pK                         (Ai) values governing the branched acid/base equilibria of Glu78 and Glu172 in BcX. These data, confirmed through theoretical calculations, help explain the pH-dependent mechanism of this model GH11 xylanase by demonstrating that the kinetically determined pK                         (A) values and hence catalytic roles of these two residues result from their electrostatic coupling.
authors.affiliation: Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. mcintosh@chem.ubc.ca
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: McIntosh
authors.forename: Daigo
authors.initials: D
authors.lastname: Naito
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Baturin
authors.forename: Mark
authors.initials: M
authors.lastname: Okon
authors.forename: Manish D
authors.initials: MD
authors.lastname: Joshi
authors.forename: Jens E
authors.initials: JE
authors.lastname: Nielsen
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D055162:Biocatalysis; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D055672:Static Electricity
chemical_list: D001426:Bacterial Proteins; D018698:Glutamic Acid; D043364:Endo-1,4-beta Xylanases