title: Characterization of a buried neutral histidine in Bacillus circulans xylanase: internal dynamics and interaction with a bound water molecule.
abstract: NMR spectroscopy was used to characterize the dynamic behavior of His149 in Bacillus circulans xylanase (BCX) and its interaction with an internal water molecule. Rate constants for the specific acid- and base-catalyzed exchange following bimolecular kinetics (EX2) of the nitrogen-bonded H epsilon 2 of this buried, neutral histidine were determined. At pDmin 7.0 and 30 degrees C, the lifetime for this proton is 9.9 h, corresponding to a protection factor of approximately 10(7) relative to that predicted for an exposed histidine. The apparent activation energies measured for specific acid and base catalysis (7.0 and 17.4 kcal/mol) indicate that exchange occurs via local structural fluctuations. Consistent with its buried environment, the N epsilon 2-H bond vector of His149 shows restricted mobility, as evidenced by an order parameter S2 = 0.83 determined from 15N relaxation measurements. The crystal structure of BCX reveals that a conserved, buried water hydrogen-bonds to the H epsilon 2 of His149. Strong support for this interaction in solution is provided by the observation of a negative nuclear Overhauser effect (NOE) and positive rotating-frame Overhauser effect (ROE) between His149 H epsilon 2 and a water molecule with the same chemical shift as the bulk solvent. However, the chemical shift of H epsilon 2 (12.2 ppm) and a D/H fractionation factor close to unity (0.89 +/- 0.02) indicate that this is not a so-called low-barrier hydrogen bond. Lower and upper bounds on the lifetime of the internal water are estimated to be 10(-8) and 10(-3) s. Therefore the chemical exchange of solvent protons with those of His149 H epsilon 2 and the diffusion or physical exchange of the internal water to which the histidine is hydrogen-bonded differ in rate by over 7 orders of magnitude.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
authors.forename: G P
authors.initials: GP
authors.lastname: Connelly
authors.forename: L P
authors.initials: LP
authors.lastname: McIntosh
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D003903:Deuterium; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D006878:Hydroxides / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D011485:Protein Binding; D012694:Serine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014867:Water / Q000378:metabolism*; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006878:Hydroxides; D009587:Nitrogen Isotopes; D014867:Water; D014443:Tyrosine; D012694:Serine; D006639:Histidine; D006859:Hydrogen; C031356:hydroxide ion; D003903:Deuterium; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase